TWI615405B - Il-1 binding proteins - Google Patents

Abstract

The present invention describes proteins that bind IL-1α and IL-1β, and their use in the treatment, prevention, and diagnosis of IL-1 related disorders and in the detection of cells, tissues, samples, and compositions of IL-1α and IL- Use of 1β composition and method.

Description

IL-1 binding protein

The present invention relates to IL-1 binding proteins and, in particular, to their use in the prevention and / or treatment of acute and rheumatoid arthritis, osteoarthritis, psoriasis, multiple sclerosis and other autoimmune diseases. Use for chronic immune disease.

Cross-reference to related applications

This application claims the priority right of US Provisional Application No. 61 / 334,917 filed on May 14, 2010 and US Provisional Application No. 61 / 425,701 filed on December 21, 2010. The content of these documents is Incorporated herein by reference in its entirety.

Cytokines such as interleukin-1 (IL-1) and tumor necrosis factor (TNF) are molecules produced by various cells such as monocytes and macrophages, which are mediators of the inflammatory process. Interleukin-1 is a cytokine with a wide range of biological and physiological effects, including fever, prostaglandin synthesis (in, for example, fibroblasts, muscle cells, and endothelial cells), T lymphocyte activation, and interleukin-2 produce.

The original members of the IL-1 superfamily are IL-1α, IL-1β, and IL-1 receptor antagonists (IL-1Ra, IL-1RA, IL-1ra, IL-1Rα). IL-1α and IL-1β are pro-inflammatory cytokines involved in immune defense against infection. IL-1Rα is a molecule that competes with IL-1α and IL-1β for binding to the receptor, thereby blocking the role of IL-1α and IL-1β in immune activation. In recent years, other molecules have been added to the IL-1 superfamily, including IL-18 (see Dinarello et al., FASEB J., 8 (15): 1314-3225 (1994); Huising et al., Dev . Comp. Immunol . , 28 (5): 395-413 (2004)) and six other genes with structural homology to IL-1α, IL-1β or IL-1RA. The last six members are named IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, and IL1F10. Accordingly, IL-1α, IL-1β, and IL-1RA have been renamed IL-1F1, IL-1F2, and IL-1F3, respectively (see Sims et al., Trends Immunol. , 22 (10): 536-537 (2001) ; Dunn et al., Trends Immunol. , 22 (10): 533-536 (2001)). Another putative member of the IL-1 family called IL-33 or IL-1F11 has been described, but this name has not been formally accepted by the HGNC gene family naming database.

IL-1α and IL-1β are produced by macrophages, monocytes and dendritic cells. It forms an important part of the inflammatory response to infection in the body. These cytokines increase the expression of adhesion factors on endothelial cells so that white blood cells that fight pathogens can be transferred to the site of infection and reset the hypothalamic temperature regulation center, thereby raising body temperature and manifesting itself as a fever. Therefore, IL-1 is called endogenous pyrogen. Increased body temperature helps the body's immune system fight infection. IL-1 is also important in regulating blood cell production. The production of IL-1β in peripheral tissues is also associated with fever-related hyperalgesia (increased sensitivity to pain) (Morgan et al., Brain Res., 1022 (1-2): 96-100 (2004)). In most cases, these two forms of IL-1 bind to the same cellular receptor. This receptor consists of two related, but not identical, subunits that transmit intracellular signals via a path that is commonly shared with some other receptors. Such receptors include the innate immune receptor and the IL-18 receptor of the Toll family. IL-1α and IL-1β also have similar biological properties, including the induction of fever, slow wave sleep and neutrophil increase, activation of T lymphocytes and B lymphocytes, proliferation of fibroblasts, and cytotoxicity to certain cells. Induces collagenase, synthesizes acute liver protein, and increases community stimulating factors and collagen production.

CDNAs encoding two different forms of IL-1 have been isolated and expressed; these cDNAs represent two different gene products called IL-1β (Auron et al., Proc. Natl. Acad. Sci. USA, 81: 7907- 7911 (1984)) and IL-1α (Lomedico et al., Nature, 312: 458-462 (1984)). IL-1β is the main form produced by human monocytes at the mRNA and protein levels. Both forms of human IL-1 share only 26% amino acid homology. Although their polypeptide sequences are different, the two forms of IL-1 have structural similarity (Auron et al., J. Mol. Cell Immunol. , 2: 169-177 (1985)), and their similarity lies in amino acid homology Sexuality is limited to individual regions of the IL-1 molecule.

IL-1α and IL-1β are produced as precursor peptides. In other words, it is prepared as a long protein and then processed to release shorter active molecules, which are called mature proteins. For example, pro-IL-1β releases mature IL after being cleaved by a member of the protein's caspase family (known as Cassin-1 or interleukin-1 converting enzyme (ICE)). -1β. The three-dimensional structure of each human IL-1 superfamily member in mature form consists of 12-14 beta strands producing barrel proteins.

Although a number of antibodies to IL-1 have been described in the last two decades of research since the discovery of this key pro-inflammatory cytokine, there remains a need for IL-1 to be effective in mediating or neutralizing inflammatory responses and autoimmune disorders Improved activity antibodies are needed to detect IL-1β in samples and tissues.

The present invention relates to proteins that bind human IL-1α and IL-1β. The binding proteins of the present invention include, but are not limited to, antibodies capable of binding human IL-1α and IL-1β, their antigen-binding portions, and multivalent, multispecific binding proteins, such as DVD-Ig ^™ binding protein. The present invention also provides methods for preparing and using the IL-1α and IL-1β binding proteins described herein, as well as methods or treatments or preventions that can be used to detect IL-1α and IL-1β in a sample and are related to IL-1 activity. Or various compositions in a method of an individual disorder suspected of being associated with IL-1 activity.

In one embodiment, the present invention provides a binding protein comprising a first and a second polypeptide chain, wherein the first polypeptide chain comprises a first VD1- (X1) n-VD2-C- (X2) n, wherein : VD1 is the first heavy chain variable domain; VD2 is the second heavy chain variable domain; C is the heavy chain constant domain; X1 is a linker, with the limitation that it is not CH1; X2 is an Fc region; and n is independently 0 or 1; and wherein the second polypeptide chain includes a second VD1- (X1) n-VD2-C- ( X2) n, where: VD1 is the first light chain variable domain; VD2 is the second light chain variable domain; C is the light chain constant domain; X1 is a linker, and the restriction is that it is not CH1; X2 does not contain Fc And n is independently 0 or 1; wherein, in the first polypeptide chain, VD1 comprises a group selected from the group consisting of SEQ ID NOs: 60-148, 196, 198, 200, 202, 204, 206, 208, and 210 And VD2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 213 and 227; wherein, in the second polypeptide chain, VD1 comprises an amino acid sequence selected from SEQ ID NO: 149- 189, 197, 199, 201, 203, 205, 207, 209, and 211; and VD2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 216 and 229; and wherein The binding protein binds human IL-1β and human IL-1α.

In one embodiment, the binding protein comprises a first polypeptide chain including an amino acid sequence selected from the group consisting of SEQ ID NOs: 212, 217, 226, 230, 232, 234, and 236.

In another embodiment, the aforementioned binding protein comprises a second polypeptide chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 215, 218, 228, 231, 233, 235, and 237.

In another aspect of the invention, the binding protein comprises a first and a second polypeptide chain, wherein The first polypeptide chain includes a first VD1- (X1) n-VD2-C- (X2) n, wherein: VD1 is a first heavy chain variable domain; VD2 is a second heavy chain variable domain; C is a heavy chain Constant domain; X1 is a linker, the restriction is that it is not CH1; X2 is an Fc region; and n is independently 0 or 1; and wherein the second polypeptide chain comprises a second VD1- (X1) n-VD2- C- (X2) n, where: VD1 is the first light chain variable domain; VD2 is the second light chain variable domain; C is the light chain constant domain; X1 is the linker, and the restriction is that it is not CH1; X2 Does not include an Fc region; and n is independently 0 or 1; wherein, in the first polypeptide chain, VD1 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 213 and 227; and VD2 includes an amino acid sequence selected from SEQ ID NO: amino acid sequence of the group consisting of 60-148, 196, 198, 200, 202, 204, 206, 208, and 210; wherein, in the second polypeptide chain, VD1 comprises a group selected from the group consisting of SEQ ID NO : An amino acid sequence of the group consisting of 216 and 229; and VD2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 149-189, 197, 199, 201, 203, 205, 207, 209, and 211; And the binding protein binds human IL-1β and human IL-1α.

In another embodiment, the binding protein comprises a first polypeptide chain including an amino acid sequence selected from the group consisting of SEQ ID NOs: 219 and 221.

In another embodiment, the aforementioned binding protein comprises a second polypeptide chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 220 and 222.

In another aspect, the present invention provides a binding protein as described above, wherein when the first polypeptide chain comprises an amino acid sequence SEQ ID NO: 212, the second polypeptide chain comprises SEQ ID NO: amino acid sequence of the group consisting of 215, 228, 231, 233, and 235; when the first polypeptide chain includes the amino acid sequence SEQ ID NO: 217, the second polypeptide chain includes the selected The amino acid sequence of the group consisting of SEQ ID NO: 218 and 237 is free; when the first polypeptide chain includes the amino acid sequence SEQ ID NO: 219, the second polypeptide chain includes the amino acid sequence SEQ ID NO: 220; when the first polypeptide chain includes an amino acid sequence SEQ ID NO: 221, the second polypeptide chain includes SEQ ID NO: 222; when the first polypeptide chain includes an amino acid sequence SEQ When ID NO: 226, the second polypeptide chain includes an amino acid sequence selected from the group consisting of SEQ ID NO: 228, 215, 231, 233, and 235; when the first polypeptide chain includes an amino acid sequence When SEQ ID NO: 230, the second polypeptide chain includes an amino acid sequence selected from the group consisting of SEQ ID NO: 231, 215, 228, 233, and 235; when the first polypeptide chain When the amino acid sequence is SEQ ID NO: 232, the second polypeptide chain includes an amino acid sequence selected from the group consisting of SEQ ID NO: 233, 215, 228, 231, and 235; when the first polypeptide When the chain includes an amino acid sequence of SEQ ID NO: 234, the second polypeptide chain includes an amino acid sequence selected from the group consisting of SEQ ID NO: 235, 215, 228, 231, and 233; and when the first When the polypeptide chain includes an amino acid sequence of SEQ ID NO: 236, the second polypeptide chain includes SEQ ID NO: 237.

In another aspect, the present invention provides a protein as described above, wherein the first polypeptide chain comprises SEQ ID NO: 212 and the second polypeptide chain comprises SEQ ID NO: 215; or the first polypeptide chain comprises SEQ ID NO: 217, and the second polypeptide chain comprises SEQ ID NO: 218; or the first polypeptide chain comprises SEQ ID NO: 219, and the second The polypeptide chain comprises SEQ ID NO: 220; or the first polypeptide chain comprises SEQ ID NO: 221 and the second polypeptide chain comprises SEQ ID NO: 222; or the first polypeptide chain comprises SEQ ID NO: 226, and the second polypeptide chain comprises SEQ ID NO: 228; or the first polypeptide chain comprises SEQ ID NO: 230, and the second polypeptide chain comprises SEQ ID NO: 231; or the first polypeptide The chain comprises SEQ ID NO: 232, and the second polypeptide chain comprises SEQ ID NO: 233; or the first polypeptide chain comprises SEQ ID NO: 234, and the second polypeptide chain comprises SEQ ID NO: 235; Or the first polypeptide chain comprises SEQ ID NO: 236 and the second polypeptide chain comprises SEQ ID NO: 237.

In one embodiment, the binding protein of the present invention described above comprises two first polypeptide chains and two second polypeptide chains.

In another aspect, in the above binding protein, X1 or X2 is an amino acid sequence selected from the group consisting of SEQ ID NOs: 26-57, 233, 224, and 225.

In another embodiment, the present invention provides a binding protein as described above, wherein the Fc region is selected from the group consisting of a natural sequence Fc region and a variant sequence Fc region. In another embodiment, the Fc region is selected from the group consisting of Fc regions from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

In another embodiment, the present invention provides a binding protein conjugate comprising the above binding protein and further comprising an agent. Such agents include, but are not limited to, immunoadhesion molecules, imaging agents, therapeutic agents, and cytotoxic agents. Preferred developers include, but are not limited to, radioactive labels, enzymes, fluorescent labels, luminescent labels, bioluminescent labels, magnetic labels, and biotin. Preferred radiolabels include, but are not limited to, ³ H, ¹⁴ C, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I, ¹⁷⁷ Lu, ¹⁶⁶ Ho, and ¹⁵³ Sm. Preferred therapeutic or cytotoxic agents include, but are not limited to, antimetabolites, alkylating agents, antibiotics, growth factors, cytokines, antiangiogenic agents, antimitotic agents, anthracycline, toxins, and cells Apoptotic agent.

The invention also provides a binding protein comprising an antigen binding domain, wherein the binding protein is capable of binding human IL-1β and the antigen binding domain comprises 6 CDRs, that is, CDR-H1, CDR-H2, CDR-H3, CDR- L1, CDR-L2 and CDR-L3, as defined below: CDR-H1: X ₁ -YDMS (SEQ ID NO: 190), where: X ₁ is S, K or R; CDR-H2: YX ₂ -SX ₄ -GGX ₇ -GTYYPDX ₁₄ -X ₁₅ -KG (SEQ ID NO: 191), where: X ₂ is I or V; X ₄ is S or H; X ₇ is G or A; X ₁₄ is T or S; And X ₁₅ is V or A; CDR-H3: GGVX ₄ -KGX ₇ -FDX ₁₀ (SEQ ID NO: 192), where: X ₄ is T or Y; X ₇ is Y or C; and X ₁₀ is V, E, L, M, Q or Y; CDR-L1: RASGNIX ₇ -X ₈ -X ₉ -LX ₁₁ (SEQ ID NO: 193), where: X ₇ is H, Y or W; X ₈ is N, G , T, Q, E, H, D or K; X ₉ is Y or W; and X ₁₁ is T, A or N; CDR-L2: X ₁ -AKX ₄ -LX ₆ -X ₇ (SEQ ID NO: 194), wherein: X ₁ is N, Q, or D; X ₄ is T, N, I, E, or S; X ₆ is A, M, or E; and X ₇ is D, E, S, or A; and CDR -L3: QX ₂ -FWX ₅ -X ₆ -PX ₈ -X ₉ (SEQ ID NO: 195), where: X ₂ is H or Q; X ₅ is S, N, T, K, R or M; X ₆ is I or L; X ₈ is Y or A; and X ₉ is T, I, and N; the exception is when CDR-H1 is SYDMS (SEQ ID NO: 17), then: CDR-H2 cannot be YISSGGGGTYYPDTVKG (SEQ ID NO: 18) CDR-H3 cannot be GGVTKGYFDV (SEQ ID NO: 19); CDR-L1 cannot be RASGNIHNYLT (SEQ ID NO: 20); CDR-L2 cannot be NAKTLAD (SEQ ID NO: 21); and CDR-L3 cannot be QHFWSIPYT (SEQ ID NO: 22).

In one embodiment, the isolated binding protein described above comprises at least one CDR comprising an amino acid sequence selected from the group consisting of a CDR sequence consisting of:

In another embodiment, the above binding protein comprises at least three CDRs, wherein the three CDRs are from a CDR set selected from the group consisting of a CDR set consisting of:

In one embodiment, the binding protein comprises CDRs from two CDR sets selected from the group of the CDR sets.

In another embodiment, the present invention provides a binding protein comprising CDRs from two sets of CDRs, wherein the two CDR sets are selected from the group consisting of:

In another embodiment, the aforementioned binding protein further comprises a human acceptor framework. The human framework preferably comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-10, 13-16, 25, 240-316, and 317-381. In one embodiment, the binding protein of the present invention comprises a human framework sequence selected from the group consisting of SEQ ID NOs: 7-10 and 13-16.

The binding protein of the present invention includes a binding protein comprising a human acceptor framework comprising at least one framework region amino acid substitution, wherein the amino acid sequence of the framework and the sequence of the human acceptor framework At least 65% identical and contain at least 70 amino acid residues identical to the framework of the human acceptor.

In another embodiment, the binding protein of the invention comprises a human acceptor framework, wherein the acceptor framework comprises at least one framework region amino acid substitution at a key residue, the key residue being selected from the group consisting of: and Residues adjacent to CDRs; residues at glycosylation sites; rare residues; residues capable of interacting with human IL-1β; residues capable of interacting with CDRs; typical residues; variable between heavy chains Contact residues between the variable region and the light chain variable region; residues in the Vernier zone; and regions that overlap between the variable heavy chain CDR1 defined by Chothia and the first heavy chain framework defined by Kabat Residues. In an exemplary embodiment, the binding protein of the present invention comprises a key residue, wherein the key residue is selected from the group consisting of 2H, 4H, 24H, 26H, 27H, 29H, 34H, 35H, 37H, 39H, 44H, 45H, 47H, 48H, 49H, 50H, 51H, 58H, 59H, 60H, 63H, 67H, 69H, 71H, 73H, 76H, 78H, 91H, 93H, 94H, 2L, 4L, 25L, 29L, 27bL, 33L, 34L, 36L, 38L, 43L, 44L, 46L, 47L, 48L, 49L, 55L, 58L, 62L, 64L, 71L, 87L, 89L, 90L, 91L, 94L, 95L (all Kabat numbers). Exemplary subsets of these residues for humanizing the binding proteins of the invention are 27H, 48H, 67H, 69H, 93H, 36L, 43L, 46L, 47L, 49L, 58L, 71L and 87L.

In another embodiment, a binding protein of the invention comprises a common human variable domain.

In one embodiment, the IL-1β binding protein of the present invention comprises at least one variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 60-189.

In another embodiment, a binding protein of the invention comprises at least one variable heavy chain (VH) region (or domain) comprising a member selected from the group consisting of SEQ ID NOs: 60-148, 196, 198, 200, 202, 204, Amino acid sequences of the group consisting of 206, 208 and 210.

In another embodiment, a binding protein of the invention comprises at least one variable light chain (VL) region (or domain) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 149-189.

In another embodiment, a binding protein of the invention comprises at least one VH region comprising an amine selected from the group consisting of SEQ ID NOs: 60-148, 196, 198, 200, 202, 204, 206, 208, and 210 Amino acid sequence; and at least one VL region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 149-189, 197, 199, 201, 203, 205, 207, 209, and 211.

In one embodiment, the binding protein of the present invention comprises two variable domains, wherein the two variable domains comprise an amino acid sequence selected from the group consisting of:

In another embodiment, the IL-1β binding protein described herein is selected from the group consisting of: an immunoglobulin molecule, scFv, a monoclonal antibody, a humanized antibody, a chimeric antibody, a humanized antibody, a Fab fragment, Fab 'fragment, F (ab') ₂ , Fv and Fv linked by disulfide bonds.

In one aspect of the invention, the binding proteins described herein are capable of modulating the biological function of IL-1. In another aspect, the binding proteins described herein are capable of neutralizing IL-1.

In one embodiment, the binding protein described herein has an on rate constant (K _on ) selected from the group consisting of: at least about 10 ² M ^-1 s ^-1 ; 10 ³ M ^-1 s ^-1 ; at least about 10 ⁴ M ^-1 s ^-1 ; at least about 10 ⁵ M ^-1 s ^-1 ; and at least about 10 ⁶ M ^-1 s ^-1 ; as resonated by surface plasmons Measured.

In another embodiment, the solutions herein have the binding protein is selected from the group consisting of the dissociation rate constant _(off rate constant, K off) of IL-1β: at most about 10 ^-3 s ^-1; at most about 10 ^{- 4} s ^-1 ; at most about 10 ^-5 s ^-1 ; and at most about 10 ^-6 s ^-1 , as measured by surface plasmon resonance.

In another embodiment, the binding protein described herein having the IL-1β solution selected from the group consisting of a dissociation constant (K _D): at most about 10 ^-7 M; at most about 10 ^-8 M; at most about 10 ^{- 9} M; at most about 10 ^-10 M; at most about 10 ^-11 M; at most about 10 ^-12 M; and at most 10 ^-13 M.

In one aspect, the invention provides a binding protein construct comprising a binding protein described herein and further comprising a linker or an immunoglobulin constant domain. In one embodiment, the binding protein construct comprises a binding protein, wherein the binding protein is selected from the group consisting of: an immunoglobulin molecule, a disulfide-linked Fv, a monoclonal antibody, scFv, a chimeric antibody, a CDR graft Antibodies, diabody, humanized antibodies, multispecific antibodies, Fab, bispecific antibodies, Fab ', bispecific antibodies, and F (ab') ₂ , DVD-Ig ^TM and Fv.

In one embodiment, the binding protein construct comprises a heavy chain immunoglobulin constant domain selected from the group consisting of: a human IgM constant domain, a human IgG4 constant domain, a human IgG1 constant domain, a human IgE constant domain, and a human IgG2 constant domain. , And human IgG3 constant domain, and human IgA constant domain.

In another embodiment, the binding protein construct comprises an immunoglobulin having an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 Constant domain.

The present invention also provides a binding protein conjugate comprising the binding protein construct described herein and further comprising an agent selected from the group consisting of an immunoadhesion molecule, a developing agent, a therapeutic agent, and a cytotoxic agent. Developers that can be used as the pharmaceutical moiety in the binding protein conjugates described herein include, but are not limited to, radioactive labels, enzymes, fluorescent labels, luminescent labels, bioluminescent labels, magnetic labels, and biotin. In one embodiment, the radioactive label is selected from the group consisting of: ³ H, ¹⁴ C, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I, ¹⁷⁷ Lu, ¹⁶⁶ Ho, and ¹⁵³ Sm.

In another embodiment, the binding protein conjugate comprises an agent as a therapeutic agent or a cytotoxic agent, which is selected from the group consisting of an antimetabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine, and an antiangiogenesis Agents, anti-mitotic agents, anthracyclines, toxins and apoptotic agents.

In one embodiment, the binding protein, binding protein construct, or binding protein conjugate described herein has a human glycosylation pattern.

The binding proteins, binding protein constructs, and binding protein conjugates described herein may exist in the form of soluble proteins or crystals. In one embodiment, the crystals are carrier-free controlled pharmaceutical release crystals. In another embodiment, the crystalline form of a binding protein, binding protein construct, or binding protein conjugate described herein has a longer in vivo half-life than its soluble counterpart. In another embodiment, the binding protein, binding protein structure described herein The crystals of the construct or binding protein conjugate retain the biological activity of their soluble counterparts.

The composition of the present invention includes a composition for releasing a crystalline binding protein, a binding protein construct, or a binding protein conjugate described herein, comprising: (a) a formulation, wherein the formulation comprises the crystals described herein A binding protein, a binding protein construct or binding protein conjugate, and a component; and (b) at least one polymeric carrier.

Polymeric carriers that can be used in the compositions of the present invention include, but are not limited to, one or more of the group consisting of: poly (acrylic acid), poly (cyanoacrylate), poly (amino acid), poly (Anhydride), poly (peptide), poly (ester), poly (lactic acid), poly (lactic-co-glycolic acid) or PLGA, poly (β-hydroxybutyrate), poly (caprolactone), poly (Dioxane), poly (ethylene glycol), poly ((hydroxypropyl) methacrylamide), poly [(organic) phosphazene], poly (orthoester), poly ( (Vinyl alcohol), poly (vinyl pyrrolidone), maleic anhydride-alkyl vinyl ether copolymer, pluronic polyols, albumin, alginate, cellulose and cellulose derivatives , Collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glucosaminoglycans, sulfated polysaccharides, blends and copolymers thereof.

In another aspect, the composition of the composition of the present invention is selected from the group consisting of albumin, sucrose, trehalose, lactitol, gelatin, hydroxypropyl-β-cyclodextrin, methoxypolyethylene glycol, and polyethylene glycol. Group of alcohols.

The invention also provides a pharmaceutical composition comprising a binding protein, a binding protein construct or a binding protein conjugate described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention may further include at least one other agent. In one embodiment, the other agents include, but are not limited to, therapeutic agents, imaging agents, cytotoxic agents, angiogenesis inhibitors; kinase inhibitors; costimulatory molecular blockers; adhesion molecule blockers; anti-cells Hormone antibodies or functional fragments thereof; methotrexate; cyclosporin; rapamycin; FK506; detectable markers or reporter conductors; TNF antagonists; antirheumatic drugs; muscle relaxation Agents, anesthetics, non-steroidal anti-inflammatory drugs (NSAIDs), analgesics Agents, anesthetics, sedatives, local anaesthetics, neuromuscular blockers, antimicrobials, anti-psoriasis drugs, corticosteroids, anabolic steroids, erythropoietin, immunoization, immunoglobulins, immunosuppressants, Growth hormones, hormone replacement drugs, radiopharmaceuticals, antidepressants, antipsychotics, stimulants, asthma drugs, beta agonists, inhaled steroids, epinephrine or similar, cytokines and cytokine antagonists.

In one embodiment, the pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier, wherein the carrier also serves as an adjuvant that increases the absorption or dispersion of binding proteins, binding protein constructs, or binding protein conjugates in the composition. Agent. An exemplary adjuvant is hyaluronidase.

In another embodiment, the pharmaceutical composition further comprises at least one other therapeutic agent for treating a condition in which IL-1β activity is detrimental.

In one embodiment, the invention provides an isolated nucleic acid encoding one or more amino acid sequences of a binding protein described herein. The nucleic acids can be inserted into a vector for various genetic analyses or used to express, characterize, or improve one or more properties of the binding proteins described herein. A vector may comprise one or more nucleic acid molecules encoding one or more amino acid sequences of a binding protein described herein, wherein the one or more nucleic acid molecules are operably linked to a suitable transcription and / or translation sequence, the Such transcription and / or translation sequences allow the binding protein to be expressed in a particular host cell having the vector. Examples of vectors used to breed or express a nucleic acid encoding an amino acid sequence of a binding protein described herein include, but are not limited to, pcDNA, pTT, pTT3, pEFBOS, pBV, pJV, and pBJ.

The invention also provides a host cell comprising a vector comprising a nucleic acid encoding one or more amino acid sequences of a binding protein described herein. The host cell that can be used in the present invention can be a prokaryotic cell or a eukaryotic cell. An exemplary prokaryotic host cell is Escherichia coli . Eukaryotic cells that can be used as host cells in the present invention include protozoan cells, animal cells, plant cells, and fungal cells. Exemplary fungal cells are yeast cells, including Saccharomyces cerevisiae . Exemplary animal cells that can be used as host cells of the invention include, but are not limited to, mammalian cells, avian cells, and insect cells. Preferred mammalian cells include CHO and COS cells. Insect cells that can be used as the host cells of the present invention are insect Sf9 cells.

In another aspect, the present invention provides a method for producing a binding protein described herein, comprising culturing in a culture medium under conditions sufficient to produce a binding protein capable of binding IL-1α and / or IL-1β comprising encoding the A host cell that binds a protein carrier. The proteins so produced can be isolated and used in various compositions and methods described herein.

In another embodiment, the present invention provides a method for reducing human IL-1 activity comprising contacting human IL-1 with a binding protein described herein to reduce human IL-1 activity.

Another embodiment of the present invention provides a method for treating an individual's condition, which is achieved by administering to the individual a binding protein described herein.

In another embodiment, the binding proteins described herein can be used to treat conditions selected from the group consisting of: diabetes; uveitis; neuralgia; osteoarthritis pain; inflammatory pain; rheumatoid arthritis; bone Arthritis; juvenile chronic arthritis; septic arthritis; Lyme arthritis; psoriasis arthritis; reactive arthritis; spinal joint disease; systemic lupus erythematosus (SLE); Crohn's disease (Crohn's disease); ulcerative colitis; inflammatory bowel disease; autoimmune diabetes; insulin-dependent diabetes; thyroiditis; asthma; allergic disease; psoriasis; dermatitis; scleroderma; graft-versus-host disease; organ transplantation Rejection; Acute immune disease related to organ transplantation; Chronic immune disease related to organ transplantation; Sarcomatoid disease; Atherosclerosis; Disseminated intravascular coagulation (DIC); Kawasaki's disease; Gray disease (Grave's disease); kidney disease syndrome; chronic fatigue syndrome; Wegener's granulomatosis (Wegener's granulomatosis); enoch-Schoenlein purpurea); renal microangiitis; chronic active hepatitis; autoimmune uveitis; septic shock; toxic shock syndrome; septic syndrome; cachexia; infectious disease; parasitic disease; acute transection Myelitis; Huntington's chorea; Parkinson's disease; Alzheimer's disease; Stroke; Primary biliary cirrhosis; Hemolytic anemia; Malignant disease; Heart failure; myocardial infarction; Addison's disease; occasional type 1 polyglandular hyposecretion; type II polyglandular hyposecretion (Schmidt's syndrome); acute respiratory distress Syndrome (ARDS); hair loss; patchy hair loss; sero-negative arthropathy; arthropathy; Reiter's disease; psoriasis arthropathy; ulcerative colon arthropathy; enteric synovitis; chlamydia ); Yarrowia sp (Yersinia) and Salmonella (Salmonella) associated arthropathy; spondyloarthropathies; atheromatous disease / arteriosclerosis; atopic allergy Autoimmune bullous disease; pemphigus vulgaris; pemphigoid pemphigoid; pemphigoid; linear IgA disease; autoimmune hemolytic anemia; Coombs positive haemolytic anaemia; acquired malignant anemia Juvenile pernicious anemia; myalgia encephalitis / Royal Free Disease; chronic mucosal skin candidiasis; giant cell arteritis (GCA); primary sclerosing hepatitis; cryptogenic autoimmune hepatitis; Acquired Immune Deficiency Syndrome (AIDS); Acquired Immune Deficiency Related Diseases; Hepatitis B; Hepatitis C; General Variant Immunodeficiency (General Variant Hypogammaglobulinemia); Dilated Cardiomyopathy; Female Infertility; Ovarian failure; Premature ovarian failure; Fibrotic lung disease; Cryptogenic fibrotic alveolitis; Post-inflammation interstitial lung disease; Interstitial pneumonia; Connective tissue disease-related interstitial lung disease; Mixed connective tissue disease-related lung disease; Systemic Sclerosis-related interstitial lung disease; rheumatoid arthritis-related interstitial lung disease; systemic lupus erythematosus-related lung disease; dermatomyositis / polymyositis-related Lung disease; Sjögren's disease-related lung disease; Ankylosing spondylitis-related lung disease; Vasculitis diffuse lung disease; Haemosiderosis-related lung disease; Drug-induced interstitial lung disease Fibrosis; Radiofibrosis; Obstructive bronchiolitis; Chronic eosinophilic pneumonia; Lymphocytic invasive lung disease; Interstitial lung disease after infection; Gouty arthritis; Autoimmune hepatitis; Type 1 autoimmune Hepatitis (typical autoimmune hepatitis or lupus hepatitis); type 2 autoimmune hepatitis (anti-LKM antibody hepatitis); autoimmune-mediated hypoglycemia; type B insulin resistance with acanthosis nigricans; parathyroid Low energy disease; Osteoarthritis; Primary sclerosing cholangitis; Type 1 psoriasis; Type 2 psoriasis; Idiopathic leukopenia; Autoimmune neutropenia; NOS nephropathy; Silky nephritis; Renal manifestation Microvasculitis; Lyme disease; Discoid lupus erythematosus; Idiopathic male infertility; Nitric oxide-related male infertility; Sperm autoimmunity; Multiple sclerosis (all subtypes, package Primary progressive, secondary progressive, relapsing-remitting); sympathetic ophthalmia; high pulmonary circulation blood pressure secondary to connective tissue disease; Goodpasture's syndrome; nodular polyarteritis Lung manifestations; acute rheumatic fever; rheumatoid spondylitis; Still's disease; systemic sclerosis; Sjörgren's syndrome; Takayasu's disease / arteritis; Autoimmune thrombocytopenia (AITP); idiopathic thrombocytopenia; autoimmune thyroid disease; hyperthyroidism; goiter autoimmune hypothyroidism (Hashimoto's disease); atrophic autoimmune Autoimmune hypothyroidism; Primary myxedema; Lens-derived uveitis; Primary vasculitis; White spot disease; Acute liver disease; Chronic liver disease; Alcoholic cirrhosis; Alcohol-induced liver injury; Cholestasis; Specific Constitutional liver disease; drug-induced hepatitis; non-alcoholic steatohepatitis; allergies; group B Streptococci (GBS) infection; mental disorders (such as suppression Depression and schizophrenia); Th2 and Th1 mediated diseases; acute and chronic pain (different forms of pain); cancer (such as lung cancer, breast cancer, stomach cancer, bladder cancer, colon cancer, pancreatic cancer, ovarian cancer, Prostate and rectal cancer); Hematopoietic malignant disease; Leukemia; Lymphoma; Abetalipoproteinemia; Acrocyanosis; Acute and chronic parasitic or infectious processes; Acute leukemia; Acute lymphoblastic leukemia ( ALL); T-cell ALL; FAB ALL; acute myeloid leukemia (AML); acute or chronic bacterial infections; acute pancreatitis; acute renal failure; adenocarcinoma; atrial ectopic beats; AIDS dementia syndrome; Alcohol-induced hepatitis; Allergic conjunctivitis; Allergic contact dermatitis; Allergic rhinitis; Allograft rejection; Alpha-1 antitrypsin deficiency; Amyotrophic lateral sclerosis; Anemia; Angina; Anterior horn cells degeneration; Anti-CD3 therapy; anti-phospholipid syndrome; anti-receptor allergic reaction; aortic and peripheral aneurysms; aortic dissection; arterial hypertension; arteriosclerosis; Arteriovenous fistula; ataxia; atrial fibrillation (persistent or paroxysmal); atrial flutter; atrioventricular block; B-cell lymphoma; bone graft rejection; bone marrow transplant (BMT) rejection; bundle Branch block; Burkitt's lymphoma; Burns; Arrhythmia; Cardiac Symptoms; Cardiac tumors; Cardiomyopathy; Cardiopulmonary bypass inflammation; Cartilage transplantation rejection; Cerebellar cortex degeneration; Cerebellar disorders; Disorders Atrial or multifocal atrial tachycardia; chemotherapy-related disorders; chronic myelogenous leukemia (CML); chronic alcoholism; chronic inflammatory lesions; chronic lymphocytic leukemia (CLL); chronic obstructive pulmonary disease (COPD); Chronic salicylate poisoning; colorectal cancer; congestive heart failure; conjunctivitis; contact dermatitis; pulmonary heart disease; coronary artery disease; Creutzfeldt-Jakob disease; culture-negative sepsis; cystic fibrosis Conditions related to cytokine therapy; boxer dementia (dementia pugilistica); demyelinating disease; hemorrhagic dengue fever; dermatitis; skin symptoms; diabetes Diabetic arteriosclerosis; diffuse Lewy body disease; dilated congestive cardiomyopathy; basal ganglia disease; middle-aged Down's syndrome; by blocking CNS dopamine receptors Drug-induced drug-induced dyskinesia; drug allergy; eczema; encephalomyelitis; endocarditis; endocrine disease; epiglottis; Epstein-Barr virus infection; erythematous limb pain; Extrapyramidal and cerebellar disorders; familial hematophagocytic lymphohistiocytosis; fetal thymus implant rejection; Friedreich's ataxia; functional peripheral arterial disorders; fungi Septicemia; gas gangrene; gastric ulcer; glomerulonephritis; transplant rejection of any organ or tissue; Gram-negative sepsis; Gram-positive sepsis; granulomas caused by intracellular organisms; hair cell leukemia; ha -Hallervorden-Spatz disease; Hashimoto's thyroiditis; hay fever; heart transplant Rejection; Hemochromatosis; Hemodialysis; Hemolytic uremic syndrome / thrombolytic thrombocytopenic purpura; Bleeding; Hepatitis A; His bundle arrhythmias; HIV infection / HIV neuropathy; Hodgkin Hodgkin's disease; hyperactive dyskinesia; allergic reaction; allergic pneumonia; hypertension; hypokinesis; hypothalamic-pituitary-adrenal axis assessment; idiopathic Addison's disease; Idiopathic pulmonary fibrosis (IPF); antibody-mediated cytotoxicity; weakness; infantile spinal muscular atrophy; aortic inflammation; influenza A; exposure to ionizing radiation; iridocyclitis / uvitis / optic neuritis Ischemia-reperfusion injury; Ischemic stroke; Juvenile rheumatoid arthritis; Juvenile spinal muscular atrophy; Kaposi's sarcoma; Kidney transplant rejection; Legionella Leishmaniasis; Leprosy; Corticospinal system lesions; Fat edema; Liver transplant rejection; Lymphedema; Malaria; Malignant lymphoma; Malignant tissue ball Multiple disease; Malignant melanoma; Meningitis; Meningococcalemia; Metabolic syndrome; Migraine; Idiopathic migraine; Mitochondrial multisystem disorder; Mixed connective tissue disease; Single plant gamma globulin disease; Multiple Myeloma; multiple system degeneration (Manche, Dejein-Thomas, Shay-Drag, and Machado-Joseph (Menzel; Dejerine-Thomas; Shy-Drager; and Machado-Joseph)); myasthenia gravis Intracellular avian mycobacterium disease; mycobacterium tuberculosis; myelodysplastic syndrome; myocardial infarction; myocardial ischemic disease; nasopharyngeal carcinoma; neonatal chronic lung disease; nephritis; renal disease; Type I muscle atrophy; neutropenic fever; non-Hodgkin's lymphoma; occlusion of the abdominal aorta and its branches; occlusive arterial disease; OKT3® therapy; testicular inflammation Periarthritis / vasterectomy; organ enlargement; osteoporosis; pancreatic transplant rejection; pancreatic cancer; tumor associated syndrome / malignant hypercalcemia; parathyroid transplant rejection; pelvic inflammation Disease; perennial rhinitis; pericardial disease; peripheral atherosclerotic disease; peripheral vascular disease; peritonitis; malignant anemia; pneumocystis carinii pneumonia; pneumonia; POEMS syndrome (polyneuropathy, organ enlargement) , Endocrine disease, single strain of gamma globulin disease and skin change syndrome); syndrome after perfusion; syndrome after pumping; syndrome after MI cardiac incision; preeclampsia; progressive supranuclear palsy; primary pulmonary hypertension; radiation therapy; Raynaud's phenomenon; Raynaud's disease; Refsum's disease; regular narrow QRS tachycardia; renal vascular hypertension; reperfusion injury; restrictive cardiomyopathy; sarcoma; Alzheimer's disease; Lewy body dementia; Seronegative arthropathy; Shock; Sickle cell anemia; Skin allograft rejection; Skin change syndrome; Small bowel transplant rejection; Solid tumor; Special arrhythmia; Spinal ataxia Imbalance; Spinal cerebellar degeneration; Streptococcus myositis; Cerebellar structural lesions; Subacute hard Panencephalitis; syncope; cardiovascular system syphilis; systemic allergies; systemic inflammatory syndrome; systemic onset juvenile rheumatoid arthritis; capillary dilatation; thromboocclusive vasculitis; thrombocytopenia; toxicity; transplantation Trauma / bleeding; type III allergic reaction; type IV allergy; unstable colic; uremia; urinary septicemia; urticaria; heart valve disease; varicose veins; vasculitis; venous disease; venous thrombosis; ventricular fiber Sexual tremor; viral and fungal infections; viral encephalitis / aseptic meningitis; virus-associated hemophagocytic syndrome; Wernicke-Korsakoff syndrome; Wilson's disease; any Organ or tissue xenograft rejection; acute coronary syndrome; acute idiopathic polyneuritis; acute inflammatory demyelinating polyradiculoneuropathy; acute ischemia; adult Still's disease; patchy alopecia; systemic Allergic reaction; Antiphospholipid antibody syndrome; Aplastic anemia; Arteriosclerosis; Atopic eczema; Atopic Inflammation; autoimmune dermatitis; autoimmune disorders associated with streptococcal infection; autoimmune bowel disease; autoimmune hearing loss; autoimmune lymphoproliferative syndrome (ALPS); autoimmune myocarditis; autoimmune premature ovarian failure Blepharitis; Bronchiectasis; Bullous pemphigoid; Cardiovascular disease; Disastrous antiphospholipid syndrome; Celiac disease; Cervical joint adhesions; Chronic ischemia; Scarring pemphigoid; Risk of multiple sclerosis Clinically isolated syndrome (CIS); conjunctivitis; childhood-onset psychosis; dacryocystitis; dermatomyositis; diabetic retinopathy; disc herniation; disc herniation; drug-induced immune hemolytic anemia; endocarditis Endometriosis; endometritis; episcleritis; erythema multiforme; severe erythema multiforme; pemphigoid gestation; Guillain-Barré syndrome (GBS); pollen fever; Hughes syndrome; idiopathic Parkinson's disease; idiopathic interstitial pneumonia; IgE-mediated allergies; immune hemolytic anemia; inclusion body myositis; infectious eyes Inflammatory diseases; inflammatory demyelinating disease; inflammatory heart disease; inflammatory nephropathy; iritis; keratitis; irritable keratoconjunctivitis; Kussmaul disease or Kussmaul-Meier disease ); Landry's paralysis; Langerhan's cell histiocytosis; Reticulum; macular degeneration; Microscopic polyangiitis; Morbus Bechterev Motor neuron disorders; Mucosal pemphigoid; Multiple organ failure; Myasthenia gravis; Bone marrow dysplasia; Myocarditis; Nerve root disorders; Neuropathy; Non-type A and non-B hepatitis; Optic neuritis; Osteolysis; Osteoarthritis JRA Peripheral arterial occlusive disease (PAOD); peripheral vascular disease (PVD); peripheral arterial disease (PAD); phlebitis; nodular polyarteritis (or nodular periarteritis); polychondritis; rheumatic polymyalgia White hair disease; polyarticular JRA; multiple endocrine deficiency syndrome; polymyositis; rheumatic polymyalgia (PMR); post-pump syndrome; primary Parkinsonism; secondary parkinsonism Prostatitis; Pure red blood cell aplasia; Primary adrenal insufficiency; Recurrent optic neuromyelitis; Restenosis; Rheumatic heart disease; SAPHO (synovitis, acne, impetigo, bone Hypertrophy and osteitis); secondary amyloidosis; shock lung; scleritis; sciatica; secondary adrenal insufficiency; polysiloxane-associated connective tissue disease; Sneddon-Wilkinson dermatosis ); Ankylosing spondylitis; Stevens-Johnson syndrome (SJS); Systemic inflammatory response syndrome; Temporal arteritis; Toxoplasma retinitis; Toxic epidermal necrolysis; Transverse myelitis; TRAPS (1 Tumor necrosis factor receptor (TNFR) -related periodic syndrome); type B insulin resistance with acanthosis nigricans; type 1 allergic reaction; type 2 diabetes; urticaria; common interstitial pneumonia (UIP) Spring conjunctivitis; viral retinitis; Vogt-Koyanagi-Harada syndrome (VKH syndrome); wet macular degeneration; wound healing; Yarrow's Salmonella bacteria and related joint disease.

In another embodiment of the method of treatment described herein, the step of administering to a subject a binding protein or binding protein construct or binding protein conjugate described herein is achieved by at least one mode selected from the group consisting of: Intestinal, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraabdominal, intrasaccular, intrachondral, intraluminal, intracelial, cerebellum, intraventricular, colon, intracervix, intragastric , Intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, Intravesical, rapid injection, transvaginal, transrectal, buccal, sublingual, intranasal and percutaneous.

Another aspect of the invention is a method of treating a patient suffering from a condition that is harmful to IL-1, which comprises administering a binding protein, binding protein construct, or binding agent described herein before, at the same time or after administration of a second agent. Step of a protein conjugate, wherein the second agent is selected from the group consisting of: inhaled steroids; beta agonists; short-acting or long-acting beta agonists; leukotriene or leukotriene receptor antagonists ADVAIR IgE inhibitor Anti-IgE antibody XOLAIR phosphodiesterase inhibitor PDE4 inhibitor xanthine anticholinergic drug mast cell stabilizer cromolyn IL-4 inhibition Agents; IL-5 inhibitors; eotaxin / CCR3 inhibitors; antagonists of histamine or its receptors (including H1, H2, H3, and H4); prostaglandin D or its receptor DP1 and CRTH2 Antagonist; TNF antagonist; soluble fragment of TNF receptor; ENBREL®; TNF enzyme antagonist; TNF converting enzyme (TACE) inhibitor; muscarinic receptor antagonist; TGF-β antagonist; interferon gamma; Pirfenidone (perfenidone); chemotherapeutic agent methotrexate; leflunomide; sirolimus (rapamycin) or an analogue thereof, CCI-779; COX2 or cPLA2 inhibition Agents; NSAIDs; immunomodulators; p38 inhibitors; TPL-2, MK-2 and NFkB inhibitors; budenoside; epidermal growth factor; corticosteroids; cyclosporine; sulfasalazine ; Aminosalicylate; 6-mercaptopurine; azathioprine; metronidazole; fat oxygenase inhibitor; mesalamine; olsalazine; bar Balsalazide; antioxidants; thromboxane inhibitors; IL-1 receptor antagonists; anti-IL-1β antibodies; anti-IL-6 antibodies; growth factors; elastase inhibitors; pyridyl-imidazole compounds ; TNF, LT, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, I L-11, IL-12, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL- 24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, EMAP-II, GM-CSF, FGF or PDGF Antibodies or agonists; antibodies to CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90, or their ligands; FK506; rapamycin; mycophenolate morpholine ethyl ester ( mycophenolate mofetil); ibuprofen; prednisolone; phosphodiesterase inhibitors; adenosine agonists; antithrombotic agents; complement inhibitors; adrenaline activators; IRAK, NIK, IKK, p38 or MAP kinase inhibitors; IL-1β converting enzyme inhibitors; TNF-α converting enzyme inhibitors; T cell signaling inhibitors; metalloproteinase inhibitors; 6-mercaptopurine; angiotensin converting enzyme inhibitors; Soluble cytokine receptor; soluble p55 TNF receptor; soluble p75 TNF receptor; sIL-1RI; sIL-1RII; sIL-6R; anti-inflammatory cytokine; IL-4; IL-10; IL-11; and TGF-β .

Another aspect of the invention provides at least one IL-1 anti-idiotype antibody against at least one IL-1 binding protein described herein. The anti-idiotypic antibody includes any protein or peptide containing a molecule comprising at least a portion of an immunoglobulin molecule, such as (but not limited to) at least one CDR of its heavy or light chain or ligand binding moiety, A heavy or light chain variable region, a heavy or light chain constant region, a framework region, or any part thereof that can be incorporated into a binding protein of the invention.

The invention relates to an IL-1β binding protein that binds IL-1β, including (but not limited to) an anti-IL-1β antibody or an antigen-binding portion thereof; and a multivalent, multispecific binding that binds IL-1β and another target. Protein, such as DVD-Ig ^™ . Various aspects of the present invention relate to antibodies and antibody fragments, DVD-Ig binding proteins, and pharmaceutical compositions thereof, and nucleic acids for preparing the IL-1β binding proteins including antibodies, DVD-Ig binding proteins and fragments thereof. Recombinant expression vectors and host cells. The present invention also encompasses methods for detecting human IL-1β using the IL-1β binding protein of the present invention; inhibiting human IL-1β in vitro or in vivo; and methods for regulating gene expression.

The invention also encompasses any binding protein or antibody capable of competing with the IL-1β binding protein described herein.

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meaning commonly understood by a person of ordinary skill. The meaning and scope of these terms should be clear, however, if there is any potential ambiguity, the definition provided herein takes precedence over any dictionary or foreign definition. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In this application, the use of "or" means "and / or" unless stated otherwise. In addition, the use of the term "including" and other forms such as "included" is not limiting. Similarly, unless specifically stated otherwise, terms such as "elements" or "components" encompass elements and components containing one unit and elements and components containing more than one subunit.

In general, the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and nomenclature for protein and nucleic acid chemistry and hybridization described herein, as well as the cell and tissue culture, molecular biology, Immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization techniques are well-known and commonly used in this technology. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and described in various general references and more specific references cited and discussed throughout this specification. Enzymatic reactions and purification techniques are according to the manufacturer's instructions, as commonly accomplished in this technique or performed as described herein. The nomenclature used in the analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein, and the laboratory procedures and techniques for the analytical chemistry, synthetic organic chemistry, and medicinal chemistry described in this article are well-known and commonly used in this technology. Laboratory procedures and techniques. Use standard techniques for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and patient treatment.

In order that the invention may be more readily understood, selected terms are defined below.

The term "polypeptide" refers to any polymer chain of an amino acid. The terms "peptide" and "protein" are used interchangeably with the term polypeptide and also refer to polymer chains of amino acids. The term "polypeptide" encompasses natural or artificial proteins, protein fragments, and polypeptide analogs of protein sequences. The polypeptide may be a monomer or a polymer. Unless the context otherwise contradicts, the term "polypeptide" encompasses fragments and variants thereof (including fragments of variants). For antigenic polypeptides, fragments of the polypeptide optionally contain at least one contiguous or non-linear epitope of the polypeptide. The exact boundaries of the at least one epitope fragment can be confirmed using general skills in the art. The fragment contains at least about 5 contiguous amino acids, such as at least about 10 contiguous amino acids, at least about 15 contiguous amino acids, or at least about 20 contiguous amino acids. Polypeptide variants are as described herein.

The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that, according to its origin or derived source, is associated with its naturally related group in its natural state. They are not related; they are essentially free of other proteins from the same species; they are expressed by cells of different species; or they do not exist in nature. Therefore, a polypeptide that is chemically synthesized or synthesized in a cellular system other than a cell of natural origin will be "isolated" from its naturally related components. Proteins can also be substantially free of natural related components by isolation using protein purification techniques well known in the art.

The term "recovery" refers to a process in which a chemical substance, such as a polypeptide, is substantially free of naturally-related components by, for example, separation using protein purification techniques well known in the art.

The term "human IL-1α" (abbreviated herein as hIL-1α or IL-1α) includes pleiotropic cytokines involved in various immune responses, inflammatory processes, and blood cell production. For example, IL-1α includes human cytokines produced by activated macrophages; it stimulates thymocyte proliferation by inducing IL-2 release, B cell maturation and proliferation, and fibroblast growth factor activity. The term human IL-1α is intended to include recombinant human IL-1α (rh IL-1α), which can be prepared by standard recombinant expression methods.

The term "human IL-1β" (abbreviated herein as hIL-1β or IL-1β) includes pleiotropic cytokines involved in various immune responses, inflammatory processes, and blood cell production. The term human IL-1β includes recombinant human IL-1β (rh IL-1β) that can be prepared by standard recombinant expression methods.

The amino acid sequences of human IL-1α and IL-1β are shown in Table 1.

The term "biological activity" refers to all the inherent biological properties of IL-1 cytokines (eg, IL-1α and / or IL-1β). Biological properties of IL-1α and IL-1β include, but are not limited to, binding to the IL-1 receptor.

The terms "specifically bind" or "specifically bind" with respect to the interaction of an antibody, protein, or peptide with a second chemical substance mean that the interaction depends on the specific structure (such as an epitope or epitope) of the chemical substance. It depends; for example, antibodies generally recognize and bind to specific protein structures rather than proteins. If the antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce binding to Amount of labeled A of the antibody.

The term "antibody" broadly refers to any immunoglobulin (Ig) molecule comprising four polypeptide chains (two heavy (H) chains and two light (L) chains), or it retains the essential epitope binding characteristics of the Ig molecule Any functional fragment, mutant, variant or derivative. Such mutant, variant or derivative antibody forms are known in the art. Non-limiting examples are discussed below.

In a full-length antibody, each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain includes a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains a domain CL. The VH and VL regions can be further subdivided into hypervariable regions interspersed with more conservative regions called framework regions (FR), called complementarity determining regions (CDR). Each VH and VL is composed of three CDRs and four FRs, which are arranged from the amine end to the carboxy end in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (eg, IgG, IgE, IgM, IgD, IgA, and IgY), any kind (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclasses.

The term "Fc region" is used to define the C-terminal region of the immunoglobulin heavy chain, which can be produced by papain digestion of intact antibodies. The Fc region may be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin generally includes two constant domains (CH2 domain and CH3 domain), and optionally a CH4 domain. Substitution of amino acid residues in the Fc portion to alter antibody effector functions is known in the art (Winter et al., U.S. Pat. Nos. 5,648,260 and 5,624,821). The Fc portion of an antibody mediates several important effector functions such as cytokine induction, ADCC, phagocytosis, complement-dependent cytotoxicity (CDC), and half-life / clearance of antibodies and antigen-antibody complexes. Depending on the therapeutic goals, these effector functions may be required for therapeutic antibodies in some cases but may be unnecessary or even harmful in other cases. Certain human IgG isotypes, particularly IgG1 and IgG3, mediate ADCC and CDC via binding to FcγRs and complement C1q, respectively. Neonatal Fc receptor (FcRn) is a key component that determines the circulating half-life of antibodies. In another embodiment, at least one amino acid residue in the constant region of the antibody (eg, the Fc region of the antibody) is replaced in order to alter the effector function of the antibody. Dimerization of two identical heavy chains of an immunoglobulin is mediated by dimerization of the CH3 domain and stabilized by disulfide bonds in the hinge region (Huber et al., Nature , 264: 415-420 (1976); Thies et al., J. Mol. Biol. , 293: 67-79 (1999)). Mutation of cysteine residues in the hinge region to prevent heavy chain-heavy chain disulfide bonds will destabilize the dimerization of the CH3 domain. Residues responsible for CH3 dimerization have been identified (Dall'Acqua et al., Biochemistry , 37: 9266-9273 (1998)). Therefore, it is possible to generate a half-valent Ig. Interestingly, these monovalent semi-Ig molecules of both IgG and IgA subclasses have been found in nature (Seligmann et al., Ann. Immunol. , 129 C: 855-870 (1978); Biewenga et al., Clin . Exp Immunol. , 51: 395-400 (1983)). The stoichiometry of the FcRn: Ig Fc region has been determined to be 2: 1 (West et al., Biochemistry , 39: 9698-9708 (2000)), and the half Fc is sufficient to mediate FcRn binding (Kim et al., Eur. J. Immunol. 24: 542-548 (1994)). Mutations that disrupt the dimerization of the CH3 domain may not have a greater adverse effect on its FcRn binding. This is due to the fact that residues important for CH3 dimerization are located on the internal interface of the CH3 b sheet structure, and are responsible for FcRn binding. This area is located on the external interface of the CH2-CH3 domain. However, hemi-Ig molecules can have some advantages in tissue penetration due to their smaller size than regular antibodies. In one embodiment, at least one amino acid residue in the constant region (eg, the Fc region) of the binding protein of the present invention is replaced in order to disrupt the dimerization of the heavy chain, resulting in a semi-DVD Ig molecule. The anti-inflammatory activity of IgG depends entirely on the sialylation of the N-linked glycans of the IgG Fc fragment. The precise glycan requirements for anti-inflammatory activity have been identified so that appropriate IgG1 Fc fragments can be produced, which in turn produces fully recombinant, sialylated IgG1 Fc with greatly enhanced potency (Anthony et al., Science , 320: 373-376 (2008)).

The term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen, such as hIL-1β. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of such antibodies may also be in a bispecific, dual specific, or multispecific format; specific binding to two or more different antigens (e.g., hIL-1β and another antigen molecule such as hIL-1β and hIL-1α). Examples of binding fragments encompassed within the "antigen-binding portion" of the term antibody include (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F (ab ') ₂ fragments, It is a bivalent fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) consisting of the VL and VH domains of one arm of an antibody Fv fragments; (v) dAb fragments containing a single variable domain (Ward et al., Nature , 341: 544-546 (1989); PCT Publication No. WO 90/05144); and (vi) isolated complements Decision region (CDR). In addition, although the two domains (VL and VH) of the Fv fragment are encoded by separate genes, they can be joined by a synthetic linker using recombinant methods that enable them to be made into pairs in which the VL and VH regions form a monovalent molecule Single protein chain (referred to as single-chain Fv (scFv); see, eg, Bird et al., Science , 242: 423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci . USA , 85: 5879-5883 (1988)). These single chain antibodies are also intended to be encompassed by the "antigen-binding portion" of the term antibody. Other forms of single chain antibodies are also contemplated, such as bifunctional antibodies. Bifunctional antibodies are bivalent, bispecific antibodies in which the VH and VL domains appear on a single polypeptide chain, but they are too short to allow paired linkers between the two domains on the same chain, thereby forcing the The isodomain is paired with the complementary domain of another chain and creates two antigen-binding sites (see, eg, Holliger et al., Proc. Natl. Acad. Sci. USA , 90: 6444-6448 (1993); Poljak, RJ, Structure 2: 1121-1123 (1994)). These antibody-binding moieties are known in the art (ed. Kontermann and Dübel, Antibody Engineering (Springer-Verlag, New York, 2001), page 790 (ISBN 3-540-41354-5)). In addition, single-chain antibodies also include "linear antibodies," which contain a pair of tandem Fv segments (VH-CH1-VH-CH1) that form one of a pair of antigen-binding regions with a complementary light chain polypeptide (Zapata et al., Protein Eng. 8 (10): 1057-1062 (1995); and U.S. Patent No. 5,641,870).

The immunoglobulin constant (C) domain refers to the heavy (CH) or light (CL) constant domain. Murine and human IgG heavy and light chain constant domain amino acid sequences are known in the art.

The term "IL-1β binding protein construct" (or "binding protein construct") refers to a polypeptide comprising one or more antigen-binding portions of the invention linked to a linker polypeptide or an immunoglobulin constant domain. A "linker polypeptide" comprises two or more amino acid residues joined by peptide bonds and is used to link one or more antigen-binding moieties. Such linker polypeptides are well known in the art (see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA , 90: 6444-6448 (1993); Poljak, RJ, Structure , 2: 1121-1123 (1994)). An immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy and light chain constant domain amino acid sequences are known in the art and are presented in Table 2.

Table 2. Sequences of the human IgG heavy chain constant domain and light chain constant domain

In addition, an IL-1β binding protein, such as an antibody or an antigen-binding portion thereof, can be a larger immunoadhesive molecule formed by covalent or non-covalent association of an antibody or antigen-binding portion with one or more other proteins or peptides. a part of. Examples of such immunoadhesive molecules include the use of a streptavidin core region to generate a tetrameric scFv molecule (Kipriyanov et al., Human Antibod . Hybridomas , 6: 93-101 (1995)) and the use of cysteine residues, The peptide and C-terminal polyhistidine tag are labeled to generate a bivalent and biotin-labeled scFv molecule (Kipriyanov et al., Mol. Immunol. , 31: 1047-1058 (1994)). Antibodies such as Fab and F (ab ') ₂ fragments can be prepared from intact antibodies using conventional techniques, such as papain or pepsin digestion of the intact antibodies, respectively. In addition, antibodies, their antigen-binding portions, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques.

"Isolated antibody" means an antibody that is substantially free of other antibodies with different antigen specificities (eg, an isolated antibody that specifically binds hIL-1β is substantially free of antibodies that specifically bind non-hIL-1β antigens). However, isolated antibodies that specifically bind hIL-1β can be cross-reactive to other antigens, such as IL-1β molecules from other species. In addition, the isolated antibodies may be substantially free of other cellular material and / or chemicals.

The term "single antibody" or "mAb" refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies that make up the population are the same except for mutations that may occur naturally in small amounts. Monoclonal antibodies are highly specific against a single antigen. In addition, each mAb is directed against a single determinant on the antigen, as compared to a multiple strain antibody preparation that typically includes different antibodies directed against different determinants (antigenic determinants). The modifier "single strain" should not be understood as requiring the production of antibodies by any particular method.

The term "human antibody" includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the invention may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations induced by random or site-specific mutations in vitro or introduced by somatic mutations in vivo), such as in CDRs And especially in CDR3. However, the term "human antibody" does not include antibodies from which the CDR sequences of the germline of another mammalian species, such as a mouse, have been transplanted onto human framework sequences.

The term "recombinant human antibody" includes all human antibodies made, expressed, produced or isolated recombinantly, such as antibodies expressed using recombinant expression vectors transfected into host cells (further described in Section II C below), self-recombinant Antibodies isolated from human antibody libraries (Hoogenboom, HR, Trends Biotechnol. , 15: 62-70 (1997); Azzazy and Highsmith, Clin. Biochem. , 35: 425-445 (2002); Gavilondo and Larrick, BioTechniques , 29: 128-145 (2000); Hoogenboom and Chames, Immunol. Today, 21: 371-378 (2000)), antibodies isolated from animals (e.g., mice) transgenic for human immunoglobulin genes (see also For example, Taylor et al., Nucl. Acids Res. , 20: 6287-6295 (1992); Kellermann and Green, Curr. Opin. Biotechnol., 13: 593-597 (2002); Little et al., Immunol. Today , 21: 364-370 (2000)); or antibodies prepared, expressed, produced, or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. These recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, the recombinant human antibodies are subjected to in vitro mutation induction (or in vivo somatic mutation induction when using animals transgenic to human Ig sequences), and therefore the recombinant antibodies are The amino acid sequences of the VH and VL regions are sequences that, although derived from and related to the human germline VH and VL sequences, may not naturally exist in the human antibody germline lineage in vivo.

The term "chimeric antibody" refers to an antibody comprising heavy and light chain variable region sequences from one species and constant region sequences from another species, such as murine heavy and light chains with Variable region antibodies.

The term "CDR-grafted antibody" refers to an antibody that contains the heavy and light chain variable region sequences from one species, but the sequence of one or more of the CDR regions of VH and / or VL has been replaced by a CDR sequence of another species, such as Antibodies with murine heavy and light chain variable regions, in which one or more murine CDRs (eg, CDR3) have been replaced with human CDR sequences.

The term "CDR" refers to the complementarity determining region within the variable sequence of an antibody. There are three CDRs in each variable region of the heavy and light chains, and each variable region is designated as CDR1, CDR2, and CDR3. The term "CDR set" as used herein refers to a group of three CDRs that occur in a single variable region capable of binding an antigen. The precise boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Maryland (1987) and (1991)) not only provides a clear residue numbering system applicable to any variable region of an antibody, Moreover, precise residue boundaries defining the three CDRs are provided. These CDRs may be referred to as Kabat CDRs. Chothia and colleagues (Chothia and Lesk, J. Mol. Biol. , 196: 901-917 (1987); and Chothia et al., Nature , 342: 877-883 (1989)) found that some sub-portions within the Kabat CDR, although having significant differences in the amino acid sequence level, adopt almost the same peptide bone structure. These sub-portions are designated as L1 , L2 and L3 or H1, H2, and H3, where "L" and "H" represent the light chain region and the heavy chain region, respectively. These regions can be called Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Defined with Kabat Other boundaries of CDR overlapping CDRs have been developed by Padlan et al. ( FASEB J. , 9: 133-139 (1995)) and MacCallum et al. ( J. Mol. Biol. , 262 (5): 732-745 (1996)) Description. Other CDR boundary definitions may not strictly follow one of the above systems, but still overlap with Kabat CDR. Specific residues or groups of residues, or even the entire CDRs, do not significantly affect the prediction or experimental discovery of antigen binding and can be shortened or lengthened. Although exemplary embodiments use CDRs defined by Kabat or Chothia, the methods used herein may be Take advantage of CDRs defined under any of these systems.

The terms "Kabat number", "Kabat definition" and "kabat mark" are used interchangeably herein. These terms are generally recognized in the art to refer to amino acids that are more variable (i.e. hypervariable) in the heavy and light chain variable regions of an antibody or its antigen-binding portion than other amino acid residues Residue Numbering System (Kabat et al., Ann. NY Acad. Sci. , 190: 382-391 (1971); and Kabat et al., Sequences of Proteins of Immunological Interest , 5th Edition , USDepartment of Health and Human Services NIH Publication No. 91-3242 (1991)). For heavy chain variable regions, the hypervariable regions range from amino acid positions 31 to 35 of CDR1, amino acid positions 50 to 65 of CDR2, and amino acid positions 95 to 102 of CDR3. For the light chain variable region, the hypervariable region ranges from amino acid positions 24 to 34 of CDR1, amino acid positions 50 to 56 of CDR2, and amino acid positions 89 to 97 of CDR3.

The growth and analysis of an extensive public database of amino acid sequences of the variable heavy and light chain regions over the past two decades has made it possible to understand the typical boundaries between the framework region (FR) and CDR sequences within the variable region sequence and This allows those skilled in the art to accurately determine CDRs based on Kabat numbering, Chothia numbering, or other systems. See, eg, Martin, "Protein Sequence and Structure Analysis of Antibody Variable Domains", Chapter 31, Antibody Engineering, (eds. By Kontermann and Dübel) (Springer-Verlag, Berlin, 2001), especially pages 432-433. The following provides a useful method for determining the amino acid sequence of the Kabat CDR within the amino acid sequence of the variable heavy chain (VH) and variable light chain (VL) regions: Identifying the CDR-L1 amino acid sequence: the amine from the VL region The base terminus starts with about 24 amino acid residues; residues before the CDR-L1 sequence are often cysteine (C); residues after the CDR-L1 sequence are often tryptophan (W) residues, Usually Trp-Tyr-Gln (WYQ), but can also be Trp-Leu-Gln (WLQ), Trp-Phe-Gln (WFQ), and Trp-Tyr-Leu (WYL); usually 10 to 17 amines in length Acid residues.

Identification of CDR-L2 amino acid sequences: 16 residues are often initiated after the CDR-L1 terminus; residues before the CDR-L2 sequence are generally Ile-Tyr (IY), but can also be Val-Tyr (VY) , Ile-Lys (IK) and Ile-Phe (IF); usually 7 amino acid residues in length.

Identification of CDR-L3 amino acid sequences: 33 amino acids are often initiated after the CDR-L2 terminus; The residue before the CDR-L3 amino acid sequence is often cysteine (C); the residue after the CDR-L3 sequence is often Phe-Gly-X-Gly (FGXG) (SEQ ID NO: 11), where X is any amino acid; it is typically 7 to 11 amino acid residues in length.

Identification of CDR-H1 amino acid sequences: about 31 amino acid residues starting from the amine end of the VH region and often 9 residues after cysteine (C); residues before the CDR-H1 sequence Often Cys-XXXXXXXX (SEQ ID NO: 12), where X is any amino acid; the residue after the CDR-H1 sequence is often Trp (W), usually Trp-Val (WV), but it can also be Trp -Ile (WI) and Trp-Ala (WA); usually 5 to 7 amino acid residues in length.

Identification of CDR-H2 amino acid sequences: 15 amino acid residues are often initiated after the CDR-H1 terminus; residues before the CDR-H2 sequence are usually Leu-Glu-Trp-Ile-Gly (LEWIG) (SEQ ID NO: 23), but can also be other variations; the residues after the CDR-H2 sequence are Lys / Arg-Leu / Ile / Val / Phe / Thr / Ala-Thr / Ser / Ile / Ala (K / RL / I / V / F / T / AT / S / I / A); usually 16 to 19 amino acid residues in length.

Identification of CDR-H3 amino acid sequences: 33 amino acid residues are often initiated after the CDR-H2 terminus and often 3 amino acid residues after cysteine (C) '; CDR-H3 sequence The former residue is often Cys-XX (CXX), where X is any amino acid, usually Cys-Ala-Arg (CAR); the residue after the CDR-H3 sequence is often Trp-Gly-X-Gly ( WGXG) (SEQ ID NO: 24), where X is any amino acid; It is usually 3 to 25 amino acid residues in length.

The term "typical" residue as used herein refers to a residue in the CDR or framework that defines a specific typical CDR structure as defined in the following literature (Chothia et al. ( J. Mol. Biol. , 196: 901-917 (1987) ); And Chothia et al. ( J. Mol. Biol. , 227: 799-817 (1992)), both of which are incorporated herein by reference). According to Chothia et al., The critical part of the CDRs of many antibodies, despite their great differences at the amino acid sequence level, have almost the same peptide bone structure. Each typical structure mainly specifies a set of peptide backbone twist angles that make adjacent segments of amino acid residues form a loop.

An "affinity matured" antibody is an antibody that has one or more changes in one or more CDRs that results in an improved affinity for the target antigen of the antibody compared to a parent antibody that does not have them. Exemplary affinity matured antibodies will have a nanomole or even a picomol affinity for the target antigen. A variety of procedures for making affinity mature antibodies are known in the art. For example, Marks et al., BioTechnology , 10: 779-783 (1992) describe affinity maturation by VH and VL domain shuffling. Barbas et al., Proc. Nat. Acad. Sci. USA , 91: 3809-3813 (1994); Schier et al., Gene , 169: 147-155 (1995); Yelton et al. , J. Immunol. , 155: 1994 -2004 (1995); Jackson et al. , J. Immunol. , 154 (7): 3310-3319 (1995); Hawkins et al., J. Mol. Biol. , 226: 889-896 (1992) describe CDRs and / Or induced by random mutation of framework residues. Selective mutations at sites of selective mutation induction and contact or hypermutation by enhanced activity of amino acid residues are described in US Patent No. 6,914,128 B1.

The term "multivalent binding protein" means a binding protein comprising two or more antigen-binding sites. Multivalent binding proteins are preferably engineered to have three or more antigen-binding sites and are generally not naturally occurring antibodies. The term "multispecific binding protein" refers to a binding protein capable of binding two or more related or unrelated targets. The "dual variable domain" ("DVD") binding protein of the present invention comprises two or more antigen binding sites and is a tetravalent or multivalent binding protein. DVD can be monospecific, that is, it can bind one Antigen; or multispecific, that is, capable of binding two or more antigens. A DVD binding protein comprising two heavy chain DVD polypeptides and two light chain DVD polypeptides is called a "DVD immunoglobulin" or "DVD-Ig". Each half of the DVD Ig contains a heavy chain DVD polypeptide and a light chain DVD polypeptide, and two or more antigen binding sites. Each binding site contains a heavy chain variable domain and a light chain variable domain. A total of 6 CDRs per antigen binding site are involved in antigen binding.

A description of the design, performance, and characterization of DVD-Ig molecules is provided in PCT Publication No. WO 2007/024715; US Patent No. 7,612,181; and Wu et al., Nature Biotechnol. , 25: 1290-1297 (2007). Preferred examples of these DVD-Ig molecules include: a heavy chain comprising the structural formula VD1- (X1) n-VD2-C- (X2) n, where VD1 is the first heavy chain variable domain and VD2 is the second heavy Chain variable domain, C is the constant domain of heavy chain, X1 is the linker, the restriction is that it is not CH1, X2 is the Fc region, and n is 0 or 1, but preferably 1; and contains the structural formula VD1- (X1 ) n-VD2-C- (X2) n light chain, where VD1 is the first light chain variable domain, VD2 is the second light chain variable domain, C is the light chain constant domain, X1 is the linker, restrictions It is not CH1, and X2 does not include an Fc region; and n is 0 or 1, but preferably 1. The DVD-Ig may include two such heavy chains and two such light chains, wherein each chain includes a variable domain connected in series, and there is no constant region inserted between the variable regions, wherein the heavy chain and the light chain associate Combined to form a tandem functional antigen binding site, and one pair of heavy and light chains can associate with another pair of heavy and light chains to form a tetrameric binding protein with four functional antigen binding sites. In another example, the DVD-Ig molecule may include a heavy chain and a light chain each including three variable domains (VD1, VD2, VD3) connected in series, with no constant regions inserted between the variable domains, one of which The heavy and light chains can be associated to form three antigen-binding sites, and one of the heavy and light chains can be associated with another pair of heavy and light chains to form a tetramer with six antigen-binding sites Binding protein.

The DVD-Ig binding protein can bind one or more epitopes of IL-1β. DVD-Ig binding protein can also bind to the epitope of IL-1β and a second target antigen other than IL-1β polypeptide Epitope.

The term "bispecific antibody" as used herein refers to a full-length antibody produced by the following technique: four-source hybridoma technology (see Milstein and Cuello, Nature , 305: 537-540 (1983)); by two Chemical conjugation of different monoclonal antibodies (see Staerz et al., Nature , 314: 628-631 (1985)); or similar by introducing a knob-into-hole in the hole or introducing mutations in the Fc region Methods (see Holliger et al., Proc. Natl. Acad. Sci. USA , 90 (14): 6444-6448 (1993)), these techniques produce multiple different immunoglobulin substances, of which only one is functionally bispecific Sexual antibodies. By molecular function, a bispecific antibody binds an antigen (or epitope) on one of its two binding arms (one pair of HC / LC) and on its second arm (the other pair of HC / LC) Binds different antigens (or epitopes). By this definition, a bispecific antibody has two different antigen-binding arms (in terms of specificity and CDR sequences) and is monovalent for each antigen to which it binds.

As used herein, the term `` dual specific antibody '' refers to a full-length antibody that can bind two different antigens (or epitopes) in each of its two binding arms (a pair of HC / LC) (see PCT Publication No. WO 02/02773). Therefore, a dual specific binding protein has two identical antigen-binding arms with the same specificity and the same CDR sequence, and is bivalent for each antigen to which it binds.

A "functional antigen-binding site" of a binding protein is a binding site capable of binding to a target antigen. The antigen-binding affinity of the antigen-binding site may not be as strong as the parent antibody from which the antigen-binding site is derived, but the ability to bind the antigen must be measurable using any of a number of methods known to assess the binding of the antibody to the antigen . In addition, the antigen-binding affinity of each antigen-binding site of a multivalent antibody herein need not be the same in number.

The term "cytokine" is a general term for proteins released by a population of cells that act as an intercellular mediator on another population of cells. Examples of such cytokines are lymphoid mediators, monocyte hormones, and conventional polypeptide hormones. Included among cytokines are growth hormones, such as human growth hormone, N-methionamine-based human growth hormone, and bovine growth hormone; Parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; pro Relaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH) and luteinizing hormone (LH); liver growth factor; Fibroblast growth factor; Prolactin; Placental lactogen; Tumor necrosis factors such as tumor necrosis factor-α (TNF-α) and tumor necrosis factor-β (TNF-β); Mullerian-inhibiting substance ); Mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-α; platelet growth factor; placental growth factor; transitional growth Factors (TGF), such as TGF-α and TGF-β; insulin-like growth factor-1 and insulin-like growth factor-11; erythropoietin (EPO); osteoinductive factors; interferons, such as interferon-α (IFN- α), interferon-β (IFN-β) and interferon-γ (IFN-γ); community stimulating factors (CSF), such as macrophage-CSF (M-CSF); granulocyte macrophage-CSF ( GM-CSF); and granulocyte-CSF (G-CSF); interleukin (IL), such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL- 17. IL-18, IL-21, IL-22, IL-23, IL-33; and other polypeptide factors, including LIF and kit ligands (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell cultures and biologically active equivalents of natural sequence cytokines.

The terms "donor" and "donor antibody" as used herein refer to an antibody that provides one or more CDRs. In an exemplary embodiment, the donor antibody is an antibody from a species different from the antibody from which the framework region was obtained or obtained. In the case of humanized antibodies, the term "donor antibody" refers to a non-human antibody that provides one or more CDRs.

The term "framework" or "framework sequence" as used herein refers to the remaining sequence of the variable region minus the CDRs. Because the precise definition of a CDR sequence can be determined by different systems, the meaning of a framework sequence can be interpreted differently accordingly. Six CDRs (CDR-L1, CDR-L2, and CDR-L3 of the light chain and CDR-H1, CDR-H2, and CDR-H3 of the heavy chain) also divide the framework regions on the light and heavy chains on each chain Four sub-regions (FR1, FR2, FR3 and FR4), of which CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. Without specifying a specific sub-region as FR1, FR2, FR3 or FR4, the framework region as mentioned refers to the combined FR in the variable region of a single naturally occurring immunoglobulin chain. As used herein, FR represents one of the four sub-regions, and FRs represents two or more of the four sub-regions constituting the framework region.

The terms "receptor" and "receptor antibody" as used herein refer to providing at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% of the amino groups of one or more framework regions An antibody to an acid sequence or a nucleic acid sequence encoding at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% of the amino acid sequence of one or more framework regions. In some embodiments, the term "receptor" refers to an antibody amino acid or a nucleic acid sequence encoding a constant region that provides a constant region. In another embodiment, the term "receptor" refers to an antibody amino acid that provides one or more of the framework and constant regions or a nucleic acid sequence encoding one or more of the framework and constant regions. In a particular embodiment, the term "receptor" refers to an amine that provides or encodes at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98%, or 100% of the amines of one or more framework regions. Human antibody amino acid or nucleic acid sequence. According to this embodiment, the receptor may contain at least one, at least two, at least three, at least four, at least five, or at least ten amino acids that do not occur at one or more specific positions of a human antibody Residues. Receptor framework regions and / or receptor constant regions can be derived, for example, from germline antibody genes, mature antibody genes, functional antibodies (e.g., antibodies well known in the art, antibodies under development, or commercially available antibodies) ).

Human heavy and light chain receptor sequences are known in the art. In one embodiment of the present invention, the human heavy and light chain receptor sequences are selected from V-base (http://vbase.mrc-cpe.cam.ac.uk/) or IMGT® (international ImMunoGeneTics information system ®) (http://imgt.cines.fr/textes/IMGTrepertoire/LocusGenes/). In another embodiment of the present invention, the human heavy and light chain receptor sequences are selected from the sequences described in Tables 3 and 4.

The term "germline antibody gene" or "gene fragment" as used herein refers to an immunoglobulin sequence encoded by a non-lymphoid cell that has not undergone a maturation process that causes genetic rearrangement and mutation for the expression of a particular immunoglobulin. (See, for example, Shapiro et al., Crit. Rev. Immunol . , 22 (3): 183-200 (2002); Marchalonis et al., Adv. Exp. Med. Biol. , 484: 13-30 (2001)). One of the advantages provided by the various embodiments of the present invention stems from the recognition that, compared to mature antibody genes, germline antibody genes are more likely to conserve the structure of basic amino acid sequences unique to individuals in a species, and therefore are therapeutic Land used in this species is unlikely to be considered to be from an alien source.

The term "critical" residue as used herein refers to certain residues in the variable region that have a greater effect on the binding specificity and / or affinity of an antibody, especially a humanized antibody. Key residues include, but are not limited to, one or more of the following: residues adjacent to the CDRs, potential glycosylation sites (may be N-glycosylation sites or O-glycosylation sites), rare Residues, residues capable of interacting with antigens, residues capable of interacting with CDRs, typical residues, contact residues between the heavy chain variable region and the light chain variable region, Residues, and residues in regions that overlap between the Chothia definition of the variable heavy chain CDR1 and the Kabat definition of the first heavy chain framework.

The term "humanized antibody" refers to sequences comprising variable regions of heavy and light chains from non-human species (e.g., mice) but in which at least a portion of the VH and / or VL sequences have been changed to more "human-like", also That is, antibodies more similar to human germline variable sequences. One class of humanized antibodies are CDR-grafted antibodies, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding non-human CDR sequences. "Humanized antibodies" are also antibodies or variants, derivatives, analogs or fragments thereof that immunospecifically bind to relevant antigens and that contain a framework (FR) region that essentially has the amino acid sequence of a human antibody and The complementarity determining region (CDR) of an amino acid sequence that is substantially non-human antibodies. The term "substantially" in the context of a CDR as used herein refers to an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or At least 99% consistent CDRs. Humanized antibodies comprise essentially all at least one and usually two variable domains (Fab, Fab ', F (ab') ₂ , FaBC, Fv), where all or substantially all CDR regions correspond to non-human immunoglobulins ( (I.e., donor antibodies) and all or substantially all of their framework regions are their framework regions common to human immunoglobulin sequences. In one embodiment, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), usually a constant region of human immunoglobulin. In some embodiments, a humanized antibody contains a light domain and at least a variable domain of a heavy chain. The antibody may also include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, a humanized antibody contains only a humanized light chain. In some embodiments, a humanized antibody contains only a humanized heavy chain. In particular embodiments, the humanized antibody contains only humanized variable domains of the light chain and / or the humanized heavy chain.

Humanized antibodies can be selected from any kind of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE; and any isotype, including (but not limited to) IgG1, IgG2, IgG3, and IgG4. Humanized antibodies can include sequences from more than one species or isotype, and specific constant domains can be selected to optimize the desired effector function using techniques well known in the art.

The framework and CDR regions of a humanized antibody need not correspond exactly to the parent sequence. For example, the donor antibody CDR or common framework can be subjected to mutagenesis by substitution, insertion and / or deletion of at least one amino acid residue, such that The CDR or framework residues at that site do not correspond to a donor antibody or a common framework. However, in an exemplary embodiment, the mutations are not extensive. Generally, at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the humanized antibody residues will correspond to those of the parental FR and CDR sequences. The term "common framework" as used herein refers to a framework region in a common immunoglobulin sequence. As used herein, the term `` co-immunoglobulin sequence '' refers to a sequence formed by amino acids (or nucleotides) that occur most frequently in related immunoglobulin sequence families (see, for example, Winnaker, From Genes to Clones (Verlagsgesellschaft) , Weinheim, Germany 1987)). In the immunoglobulin family, each position in the common sequence is occupied by the amino acid that occurs most frequently at another position in the family. If two amino acids occur The same, any one can be included in the common sequence.

With regard to the construction of DVD-Ig or other binding protein molecules, a "linker" is used to indicate a single amino acid or a polypeptide containing two or more amino acid residues linked by a peptide bond ("linker polypeptide") and It is used to attach one or more antigen-binding moieties. Such linker polypeptides are well known in the art (see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA , 90: 6444-6448 (1993); Poljak, RJ, Structure , 2: 1121-1123 ( 1994)). Exemplary linkers include, but are not limited to, GGGGSG (SEQ ID NO: 26), GGSGG (SEQ ID NO: 27), GGGGSGGGGS (SEQ ID NO: 28), GGSGGGGSG (SEQ ID NO: 223), GGSGGGGSGS (SEQ ID NO: 29), GGGGGGGSGGGGS (SEQ ID NO: 30), GGGGSGGGGSGGGG (SEQ ID NO: 31), GGGGSGGGGSGGGGS (SEQ ID NO: 32), ASTKGP (SEQ ID NO: 33), ASTKGPSVFPLAP (SEQ ID NO: 34), TVAAP (SEQ ID NO: 35), RTVAAP (SEQ ID NO: 224), TVAAPSVFIFPP (SEQ ID NO: 36), RTVAAPSVFIFPP (SEQ ID NO: 225), AKTTPKLEEGEFSEAR (SEQ ID NO: 37), AKTTPKLEEGEFSEARV (SEQ ID NO : 38), AKTTPKLGG (SEQ ID NO: 39), SAKTTPKLGG (SEQ ID NO: 40), SAKTTP (SEQ ID NO: 41), RADAAP (SEQ ID NO: 42), RADAAPTVS (SEQ ID NO: 43), RADAAAAGGPGS (SEQ ID NO: 44), RADAAAAGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 45), SAKTTPKLEEGEFSEARV (SEQ ID NO: 46), ADAAP (SEQ ID NO: 47), ADAAPTVSIFPP (SEQ ID NO: 48), QPKAAP (SEQ ID NO: 49), QPKAAPSVTLFPP (SEQ ID NO: 50), AKTTPP (SEQ ID NO: 51), AKTTPPSVTPLAP (SEQ ID NO: 52), AKTTAP (SEQ ID NO: 53), AKTTAPSVYPLAP (SEQ ID NO: 54), GENKVEYAPA LMALS (SEQ ID NO: 55), GPAKELTPLKEAKVS (SEQ ID NO: 56), and GHEAAAVMQVQYPAS (SEQ ID NO: 57).

As used herein, the "Veneer" region refers to a subset of framework residues that can adjust the structure of CDRs and fine-tune the coordination with antigens, such as Foote and Winter, J. Mol. Biol. , 224: 487-499 (1992) (which (Incorporated herein by reference). The residues of the Venier region form a layer below the CDR and have an effect on the CDR structure and antibody affinity.

The term "neutralizing" as used herein refers to the elimination of a biological activity of a binding protein when the binding protein specifically binds an antigen (eg, the cytokine IL-1β). The neutralizing binding proteins described herein preferably bind hIL-1β, resulting in inhibition of the biological activity of hIL-1β. The neutralizing binding protein preferably binds hIL-1β and reduces the biological activity of hIL-1β by at least about 20%, 40%, 60%, 80%, 85%, or 85% or more. The inhibition of hIL-1β biological activity by the neutralizing binding protein can be assessed by measuring one or more indicators of hIL-1β biological activity well known in the art. For example, in HS27 cells, IL-1β induces inhibition of human IL-6 secretion.

The term "activity" includes the activity of an antibody, such as binding specificity / affinity, to an antigen, for example, an anti-h IL-1β antibody that binds to the IL-1β antigen and / or antibody neutralizing potency, such as an anti-IL-1β antibody and h IL -1β binding inhibits the biological activity of h IL-1β, such as the inhibition of IL-1β induction on human IL-6 secretion in HS27 cells.

The term "antigenic determinant" includes any polypeptide determinant capable of specifically binding an immunoglobulin or T cell receptor. In some embodiments, the epitope determinant includes a chemically active surface group of a molecule such as an amino acid, a sugar side chain, a phosphonium group, or a sulfonyl group, and may have a specific three-dimensional structure in some embodiments Characteristics and / or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody. In certain embodiments, when an antibody preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules, it is said to specifically bind the antigen. If antibodies cross-compete (one prevents the binding or regulatory effect of the other), then the antibody is said to "bind to the same epitope." In addition, the structural definitions (overlapping, similar, and identical) of epitopes are informative, but because functional definitions cover structural (binding) and functional (regulatory, competitive) parameters, they are usually more relevant.

As used herein, the term `` surface plasmon resonance '' refers to the analysis of live organisms by detecting changes in the concentration of proteins in the biosensor matrix using, for example, the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, New Jersey). Optical phenomenon of specific interactions. For further description, see Jönsson et al., Ann. Biol. Clin. , 51: 19-26 (1993); Jönsson et al., BioTechniques , 11: 620-627 (1991); Johnson et al., J. Mol. Recognit. 8: 125-131 (1995); and Johnsons et al., Anal. Biochem. , 198: 268-277 (1991).

The term "K _on " (also "Kon", "kon") as used herein means that a binding protein (e.g., an antibody) as known in the art is associated with an antigen to form an association complex (e.g., an antibody / Antigen complex). "K _on " is also known as the term "association rate constant" or "ka", which is used interchangeably herein. This value indicates the rate of association of the antibody with its target antigen or the rate of complex formation between the antibody and the antigen, as shown by the following equation: antibody ("Ab") + antigen ("Ag") → Ab-Ag.

The term "K _off " (also "Koff", "koff") as used herein means the dissociation of a binding protein (e.g., an antibody) from an association complex (e.g., an antibody / antigen complex) as known in the art Dissociation rate constant, or "dissociation rate constant". This value indicates the dissociation rate of an antibody from its target antigen or the Ab-Ag complex as time passes, as shown by the following equation: Ab + Ag ← Ab-Ag.

The term “K _D ” (also “K _d ”) as used herein means “equilibrium dissociation constant” and refers to a value obtained in an equilibrium state in titration measurement, or by changing the dissociation rate constant (Koff ) Divided by the association rate constant (Kon). The association rate constant (Kon), dissociation rate constant (Koff), and equilibrium dissociation constant (K _D ) are used to represent the binding affinity of an antibody for an antigen. Methods for determining association and dissociation rate constants are well known in the art. Uses fluorescence-based technology to provide high sensitivity and the ability to check samples in equilibrium in physiological buffers. Other experimental methods and instruments may be used, such as BIAcore® (Biomolecular Interaction Analysis) assays (eg, instruments available from BIAcore International AB, GE Healthcare, Uppsala, Sweden). Alternatively, a KinExA® (Kinetic Exclusion Assay) assay can be used, which is available from Sapidyne Instruments (Boise, Idaho).

The terms "label" and "detectable label" mean attached to a specific binding partner (such as an antibody or an analyte), for example to make the reaction between members of a specific binding pair (such as an antibody and an analyte) detectable section. A specific binding partner (such as an antibody or analyte) so labeled is referred to as a "detectably labeled". Accordingly, the term "labeled binding protein" as used herein refers to a protein having a marker incorporated that provides identification of the binding protein. In one embodiment, the labeling is a detectable label that can generate a signal that can be detected visually or instrumentally, such as incorporating a radiolabeled amino acid or using a labeled avidin Or a biotin-based moiety detected by streptavidin (such as a streptavidin containing a fluorescent label or an enzymatically active detectable by optical or colorimetric methods) is attached to the polypeptide. Examples of polypeptide labeling include (but are not limited to) the following: radioisotopes or radionuclides (e.g. ³ H, ¹⁴ C, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I, ¹⁷⁷ Lu, ¹⁶⁶ Ho Or ¹⁵³ Sm); prochromin; fluorescent labels (such as FITC, rhodamine, lanthanide phosphors); enzyme labels (such as horseradish peroxidase, luciferase, alkaline phosphatase); Chemiluminescent labels; biotin-based; predetermined polypeptide epitopes recognized by secondary reporters (such as leucine zipper pair sequences, secondary antibody binding sites, metal binding domains, epitope tags); and magnetic Agents (such as europium chelates). Representative examples of labels commonly used in immunoassays include a light-generating moiety (e.g., an azadine compound) and a fluorescent-generating moiety (e.g., luciferin). Other tags are described in this article. In this regard, the part itself may not be detectably labeled but may become detectable after reacting with another part. The use of the term "detectable mark" is intended to cover the latter type of detectable mark.

The term "IL-1β binding protein conjugate" refers to an IL-1β binding protein described herein that is chemically linked to a second chemical moiety, such as a therapeutic or cytotoxic agent. The term "agent" is used in this context This text is used to indicate compounds, mixtures of compounds, biological macromolecules or extracts made from biological materials. Therapeutic or cytotoxic agents preferably include, but are not limited to, pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide bromide), emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine ( colchicine, cranberry (doxorubicin), daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D (actinomycin D), 1-dihydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol And puromycin and its analogs or homologs. When used in the context of an immunoassay, the IL-1β binding protein conjugate can be a detectably labeled antibody used as a detection antibody.

The terms "crystal" and "crystallized" as used herein refer to a binding protein (e.g., an antibody) or an antigen-binding portion thereof in the form of a crystal. A crystal is a form of solid matter that is different from other forms such as an amorphous solid state or a liquid crystal state. Crystals consist of a regular, repeating, three-dimensional array of atoms, ions, molecules (e.g., proteins such as antibodies) or molecular assemblies (e.g., antigen / antibody complexes). These three-dimensional arrays are arranged according to specific mathematical relationships that are well understood in the art. Basic units or building blocks that repeat in a crystal are called asymmetric units. Asymmetric units repeated in an arrangement that conforms to the established, well-defined crystallographic symmetry will provide the "unit cell" of the crystal. A unit cell will provide a crystal by repeating regular translation in all three dimensions. See Giegé et al., Chapter 1, Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd Edition , (ed. Ducruix and Giegé) (Oxford University Press, New York, 1999) pages 1-16.

The term "polynucleotide" means two or more nucleotides (ribonucleotides or A multimeric form of an oxynucleotide or a modified form of any type of nucleotide). The term includes single- and double-stranded DNA.

The term "isolated polynucleotide" means a polynucleotide (e.g., derived from a genomic, cDNA, or synthetic source, or some combination thereof), according to which source "isolated polynucleotide": and All or part of a polynucleotide found in nature with an "isolated polynucleotide" is not related; operably linked to a polynucleotide that is not linked to it in nature; or not as a larger sequence Part of it exists in nature.

The term "vector" as used herein means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop that can join other DNA segments. Another type of vector is a viral vector, where other DNA segments can be joined in the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (such as non-episomal mammalian vectors) can be integrated into the host cell's genome after introduction into the host cell, and thereby be replicated together with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. These vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors used in recombinant DNA technology are usually in the form of plastids. In this specification, because plastids are the most common form of carrier, "plasmid" and "carrier" are used interchangeably. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which provide equivalent functionality.

The term "operably linked" refers to the juxtaposition of the components in a relationship that allows them to function in their intended manner. The control sequences "operably linked" to the coding sequence are joined in a manner such that the performance of the coding sequence is achieved under conditions compatible with the control sequence. An "operably linked" sequence includes a performance control sequence adjacent to a related gene and a performance control sequence that acts on the opposite side or at a distance to control the related gene. The term "performance control sequence" as used herein refers to the performance and processing of the coding sequences to which it is joined. The necessary polynucleotide sequence. Performance control sequences include appropriate transcription initiation sequences, termination sequences, promoter sequences and enhancer sequences; effective RNA processing signals, such as splicing signals and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (also (Kozak common sequence); sequences that enhance protein stability; and sequences that enhance protein secretion when needed. The nature of these control sequences varies depending on the host organism; in prokaryotes, these control sequences generally include promoters, ribosome binding sites, and transcription termination sequences; in eukaryotes, these control sequences generally include promoters And transcription termination sequences. The term "control sequence" is intended to include components whose presence is essential for performance and processing; and may also include other components whose presence is advantageous, such as leader sequences and fusion partner sequences.

"Transformation" as defined herein refers to any process by which foreign DNA enters a host cell. Transformation can be performed under natural or artificial conditions using a variety of methods well known in the art. Transformation can rely on any known method of inserting foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the transformed host cells and may include, but is not limited to, viral infection, electroporation, liposome transfection, and particle bombardment. Such "transformed" cells include stably transformed cells whose inserted DNA is capable of replicating in the form of autonomously replicating plastids or as part of the host chromosome. It also includes cells that transiently express the inserted DNA or RNA for a limited period of time.

The term "recombinant host cell" (or simply "host cell") means a cell into which foreign DNA has been introduced. In one embodiment, the host cell comprises two or more (e.g., multiple) nucleic acids encoding antibodies, such as the host cells described in US Patent No. 7,262,028. These terms are intended to refer not only to specific individual cells, but also to the progeny of such cells. Because certain modifications may be present in the progeny due to mutations or environmental effects, the progeny may not actually be the same as the parent cell, but are still included within the scope of the term "host cell" as used herein. In one embodiment, the host cells include prokaryotic and eukaryotic cells selected from any of the biological world. In another embodiment, the eukaryotic cells include protozoa, eukaryotes, Bacteria, plant and animal cells. In another embodiment, the host cell includes (but is not limited to) a prokaryotic cell line E. coli; a mammalian cell line CHO, HEK 293, COS, NSO, SP2, and PER.C6; an insect cell line Sf9; and a fungal cell Saccharomyces cerevisiae .

Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (eg, electroporation, liposome transfection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions or as commonly accomplished in such techniques or as described herein. The above techniques and procedures can generally be performed according to conventional methods well known in the art and described in various general and more specific references that are cited and discussed throughout this specification. See, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual , 2nd Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

A "transgenic organism" as known in the art refers to an organism having a cell containing a transgene, in which a transgenic gene introduced into an organism (or an ancestor of the organism) does not behave as a polypeptide that naturally occurs in the organism. A "transgenic gene" is a DNA construct that is stably and operatively integrated into the genome of a cell that can develop into a transgenic organism, thereby directing the encoded gene product in one or more cell types of the transgenic organism Or performance in the organization.

The terms "modulate" and "modulate" are used interchangeably, and as used herein, refer to a change or change in the activity of a related molecule (eg, the biological activity of h IL-1β). Adjustment can be to increase or decrease the magnitude of a certain activity or function of a related molecule. Exemplary activities and functions of the molecule include, but are not limited to, binding properties, enzymatic activity, cellular receptor activation, and signal transduction.

Accordingly, the term "modulator" as used herein is a compound capable of altering or changing the activity or function of a related molecule, such as the biological activity of hIL-1β. For example, a modulator can increase or decrease the magnitude of a certain activity or function of a molecule compared to the magnitude of an activity or function observed in the absence of a modulator. In certain embodiments, a modulator is an inhibitor that reduces the magnitude of at least one activity or function of a molecule. Exemplary inhibitors include, but are not limited to, proteins, peptides, antibodies, peptibody, carbohydrate Compounds or small organic molecules. Peptides are described, for example, in PCT Publication No. WO 01/83525.

As used herein, the term "agonist" means that when contacted with a molecule of interest, the activity or function of a molecule is compared to the amount of activity or function observed in the absence of the agonist. Increased volume regulator. Related specific agonists may include, but are not limited to, an IL-1β polypeptide, nucleic acid, carbohydrate, or any other molecule that binds hIL-1β.

As used herein, the terms "antagonist" and "inhibitor" refer to the activity of a molecule when compared to the amount of activity or function observed in the absence of the antagonist when contacted with the molecule of interest. Or modifiers with reduced magnitude of function. Related specific antagonists include antagonists that block or modulate the biological or immune activity of human IL-1β. Antagonists and inhibitors of human IL-Ι β may include, but are not limited to, proteins, nucleic acids, carbohydrates, or any other molecule that binds human IL-Ι β.

The term "effective amount" as used herein means sufficient to reduce or improve the severity and / or duration of the condition or one or more symptoms thereof; prevent the progression of the condition; promote the regression of the condition; prevent the recurrence of one or more symptoms associated with the condition, Appearance, onset, or progression; detection of a condition; or the amount of a therapy that enhances or improves the preventive or therapeutic effect of another therapy, such as a prophylactic or therapeutic agent.

"Patient" and "individual" are used interchangeably herein to refer to animals, such as mammals, including primates (such as humans, monkeys, and chimpanzees), non-primates (such as cows, pigs, camels, Llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, whales), birds (such as ducks or geese) and sharks. Preferably, the patient or individual is a human, such as a human being treated or evaluated for a disease, disorder, or condition, a human at risk for the disease, disorder, or condition, a human having the disease, disorder, or condition, and / Or a human being treated for a disease, disorder, or condition.

The term "sample" as used herein is used in its broadest sense. As used herein, a `` biological sample '' includes, but is not limited to, any amount of a living thing or something that was originally a living thing quality. Such living things include, but are not limited to, humans, non-human primates, mice, rats, monkeys, dogs, rabbits, and other animals. Such substances include, but are not limited to, blood (eg, whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, white blood cells, monocytes, other cells, organs, tissues, bone marrow, lymph nodes, and spleen.

"Components" and "at least one component" generally refer to kits that can be included for testing a sample (such as a patient's urine, serum, or plasma sample) according to the methods described herein and other methods known in the art. Capture antibodies, detection or binding antibodies, controls, calibrators, a series of calibrators, sensitivity panels, containers, buffers, diluents, salts, enzymes, enzyme cofactors, Detection reagents, pretreatment reagents / solutions, substrates (e.g., in solution), termination solutions and the like. Therefore, in the context of the present invention, "at least one component" and "component" may include a polypeptide or other analyte as described above, such as a composition comprising an analyte (such as a polypeptide), as appropriate, such as by binding to Anti-analyte (eg, anti-polypeptide) antibodies are immobilized on a solid support. Some components may be in a dissolved state or lyophilized for reconstitution for testing.

"Control" refers to a composition that is known not to be an analyte ("negative control") or contains an analyte ("positive control"). The positive control may include a known concentration of the analyte. "Control," "positive control," and "calibrator" are used interchangeably herein to refer to a composition comprising a known concentration of an analyte. A "positive control" can be used to establish assay performance characteristics and is a suitable indicator of the integrity of a reagent (eg, an analyte).

"Predetermined cut-off value" and "predetermined degree" generally refer to the test cut-off value used to evaluate the diagnosis / prognosis / treatment efficacy result by comparing the test results against the predetermined cut-off value / degree. Parameters (such as disease severity, progression / no progression / improvement, etc.) are linked or correlated. Although this disclosure may provide an exemplary degree of presupposition, it is well known that cut-off values may vary depending on the nature of the immunoassay (e.g., antibodies used, etc.). In addition, modifying the disclosure in this article for other immunoassays to obtain specific immunoassay cutoffs for their other immunoassays based on this disclosure is entirely part of this Within the general skills of the technician. Although the exact value of the predetermined cut-off value / degree may vary from test to test, the correlation (if any) as described herein should generally be applicable.

A "pretreatment reagent" (e.g., a lysing, precipitating, and / or solubilizing reagent) used in a diagnostic assay as described herein is a cell that can dissolve and / or cause any cell to be present in a sample. Analyte solubilizing reagent. As described further herein, pre-processing is not necessary for all samples. Among them, solubilizing an analyte (eg, a related polypeptide) may require releasing the analyte from any endogenous binding protein present in the sample. The pretreatment reagent may be homogeneous (no separation step required) or heterogeneous (requires a separation step). By using a heterogeneous pretreatment reagent, any precipitated analyte-binding protein is removed from the sample and the next step in the assay is performed.

"Quality Control Reagents" in the context of the immunoassays and kits described herein include, but are not limited to, calibrators, controls, and sensitivity kits. A "calibrator" or "standard" (eg, one or more, such as a plurality of calibrators or standards) is typically used to establish a calibration (standard) curve to interpolate the concentration of an analyte, such as an antibody or analyte. Alternatively, a single calibrator close to a predetermined positive / negative cut-off value can be used. Multiple calibrators (ie, more than one calibrator or different amounts of calibrators) can be used in combination to form a "sensitivity kit".

"Risk" means the likelihood or probability that a particular event will occur now or at some point in the future. "Risk stratification" refers to a series of known clinical risk factors that enable a physician to attribute a patient to a low, medium, high, or highest risk of a particular disease, disorder, or condition.

"Specificity" in the case of an interaction between members of a specific binding pair, such as an antigen (or a fragment thereof) and an antibody (or an antigen-reactive fragment thereof), refers to the selective reactivity of the interaction. The phrase "specifically binds" and similar phrases refers to the ability of an antibody (or antigen-reactive fragment thereof) to specifically bind to an analyte (or fragment thereof) and not specifically to other entities.

A "specific binding partner" is a member of a specific binding pair. The specific binding pair comprises 2 different molecules, which specifically bind to each other via chemical or physical means. Therefore, in addition to antigen-antibody specific binding pairs of common immunoassays, other specific binding pairs may include biotin and avidin (or streptavidin); carbohydrates and lectins; complementary nucleotide sequences Effector and receptor molecules; cofactors and enzymes; enzyme inhibitors and enzymes; and the like. In addition, a specific binding pair may include a member that is an analog of the initial specific binding member, such as an analyte-analog. Immunoreactive specific binding members include isolated or recombinantly produced antigens, antigen fragments and antibodies, including single and multiple strains of antibodies and their complexes, fragments and variants (including variant fragments).

As used herein, a `` variant '' means a amino acid sequence that differs from a given polypeptide (e.g., IL-1β, BNP, NGAL, or HIV polypeptide or due to amino acid addition (e.g., insertion), deletion, or conservative substitution). Anti-polypeptide antibody), but retains the biological activity of a given polypeptide (eg, a variant IL-1β can compete with an anti-IL-1β antibody for binding to IL-1β). Conservative substitutions of amino acids (ie, replacing amino acids with different amino acids having similar properties, such as hydrophilicity and extent and distribution of charged regions) are generally considered in the art to involve minor changes. As is known in the art, these minor changes can be identified in part by considering the hydropathic index of the amino acid (see, for example, Kyte et al., J. Mol. Biol. , 157: 105-132 ( 1982)). The hydrophilic index of amino acids is based on considerations of their hydrophobicity and charge. It is known in the art that amino acids with similar hydrophilic indices can be substituted and still retain protein function. In one aspect, an amino acid having a hydrophilic index of ± 2 is substituted. The hydrophilic nature of amino acids can also be used to reveal substitutions that would produce proteins that retain biological functions. The consideration of amino acid hydrophilicity in the case of a peptide allows calculation of the maximum local average hydrophilicity of the peptide, which is a suitable measure that has been reported to be closely related to antigenicity and immunogenicity (see, for example, U.S. Patent No. 4,554,101) . As is known in the art, substitution of amino acids with similar hydrophilicity values can produce peptides that retain biological activity (e.g., immunogenicity). In one aspect, substitution is performed with amino acids having a hydrophilicity value within a range of ± 2 from each other. The amino acid's hydrophobicity index and hydrophilicity value are both affected by the specific side chain of the amino acid. Consistent with this observation, amino acid substitutions that are compatible with biological functions are understood to depend on the relative similarity of amino acids and especially their side chains, such as hydrophobicity, hydrophilicity, charge, Size and other properties revealed. A "variant" can also be used to describe a polypeptide that has been differentially processed (such as by proteolysis, phosphorylation, or other post-translational modification) but still retains its biological activity or antigenic reactivity (such as the ability to bind Its fragment. Unless the context contradicts otherwise, the term "variant" is used herein to encompass fragments of the variant.

I. Antibodies that bind human IL-1β

One aspect of the present invention provides an isolated murine monoclonal antibody or an antigen-binding portion thereof that binds to IL-1β with high affinity, slow dissociation rate, and high neutralization ability. A second aspect of the invention provides a chimeric antibody that binds IL-1β. A third aspect of the present invention provides a CDR-grafted antibody or an antigen-binding portion thereof that binds IL-1β. A fourth aspect of the present invention provides a humanized antibody or an antigen-binding portion thereof that binds IL-1β. A fifth aspect of the present invention provides a dual variable domain immunoglobulin (DVD-Ig ^™ ) molecule that binds IL-1β and another target. The antibody or part thereof is preferably an isolated antibody. The antibody of the present invention is preferably a neutralizing human anti-IL-1β antibody.

A. Methods for preparing anti-IL-1β antibodies

The anti-IL-1β antibodies of the present invention can be prepared by any of a number of techniques known in the art.

1. Production of anti-IL-1β monoclonal antibodies using fusion tumor technology

Monoclonal antibodies can be prepared using a variety of techniques known in the art, including the use of fusion tumor technology, recombinant technology, and phage presentation technology, or a combination thereof. For example, monoclonal antibodies can be produced using fusion tumor technology, including those known in the art and taught in, for example, the following references: Harlow et al., Antibodies: A Laboratory Manual , 2nd Edition (Cold Spring Harbor Laboratory Press, 1988); Hammerling et al., "Monoclonal Antibodiess and T-Cell Hybridomas", Research Monographs in Immunology , Volume 3 (Editor in Chief JLTurk) (Elsevier, New York, 1981) pp. 563-587 (the And other references are incorporated herein by reference in their entirety). The term "monoclonal antibody" as used herein is not limited to antibodies produced via fusion tumor technology. The term "single antibody" refers to an antibody derived from a single pure line (including any eukaryotic, prokaryotic or phage pure line), regardless of the method by which it is produced.

Methods for generating and screening specific anti-IL-1β antibodies using fusion tumor technology are conventional methods and are well known in the art. In one embodiment, the present invention provides a method for producing a monoclonal antibody and an antibody produced by the method, the method comprising culturing a fusion tumor cell that secretes the antibody of the present invention, wherein the fusion tumor is preferably obtained by using the antigen of the present invention The spleen cells isolated from the immunized mice are fused with myeloma cells and are subsequently produced by screening for fusion tumors produced by the fusion in a pure line of fusion tumors that secrete antibodies capable of binding to the polypeptide of the invention. Briefly, mice can be immunized with the IL-1β antigen. In an exemplary embodiment, the IL-1 β antigen is administered with an adjuvant to stimulate an immune response. Such adjuvants include complete or incomplete Freund's adjuvant, RIBI (cell wall dipeptide) or ISCOM (immunostimulatory complex). These adjuvants can protect the polypeptide from rapid dispersal by sequestering the polypeptide in local sediments, or they can contain substances that stimulate the host to secrete factors that are chemotactic to macrophages and other components of the immune system . Preferably, if the polypeptide is administered, the immunization schedule will involve the administration of the polypeptide in two or more portions over a period of several weeks.

After the animal is immunized with the IL-1β antigen, antibodies and / or antibody-producing cells can be obtained from the animal. Animals obtained serum containing anti-IL-1β antibodies by bleeding or sacrifice animals. The serum can be used as it is when the animal is obtained, the immunoglobulin fraction can be obtained from the serum, or the anti-IL-1β antibody can be purified from the serum. The serum or immunoglobulins obtained in this way are multiple strains and thus have a range of different properties.

Once an immune response is detected, such as the detection of antibodies specific for the antigen IL-1β in mouse serum, the mouse spleen is harvested and spleen cells are isolated. The spleen cells and any suitable myeloma cells (e.g. from American Type Culture Collection (ATCC, Manassas, Virginia) cell line SP20) fusion. Fusion tumors were selected and selected by limiting dilution. Cells that secrete antibodies capable of binding IL-1β are subsequently assayed for fused tumor pure lines by methods known in the art. Ascites, which typically contains high levels of antibodies, can be produced by immunizing mice with a pure line of positive fusion tumors.

In another embodiment, an antibody-producing immortalized fusion tumor can be prepared from an immunized animal. Following immunization, the animals are sacrificed and the splenic B cells are fused with immortalized myeloma cells as is well known in the art. See, eg, Harlow et al., Supra. In an exemplary embodiment, the myeloma cells do not secrete an immunoglobulin polypeptide (a non-secretory cell line). After fusion and antibiotic selection, fused tumors are screened using IL-1β or a portion thereof or cells expressing IL-1β. In an exemplary embodiment, the initial screening is performed using an enzyme-linked immunoassay (ELISA) or a radioimmunoassay (RIA), preferably an ELISA. An example of an ELISA screen is provided in PCT Publication No. WO 00/37504, which is incorporated herein by reference.

Fusion tumors producing anti-IL-1β antibodies are selected, colonized, and further screened for desired characteristics, including stable fusion tumor growth, high antibody production, and desired antibody characteristics as discussed further below. Fusion tumors can be cultured and expanded in vivo in animals of the same genotype, animals lacking the immune system (such as nude mice), or cultured and expanded in vitro in cell cultures. Methods for selecting, colonizing and expanding fusion tumors are well known to those of ordinary skill.

In an exemplary embodiment, the fusion tumor is a mouse fusion tumor as described above. In another preferred embodiment, the fusion tumor line is produced in a non-human, non-mouse species, such as a rat, sheep, pig, goat, cow, or horse. In another embodiment, the fusion tumor is a human fusion tumor, wherein a human non-secretory myeloma is fused with a human cell expressing an anti-IL-1β antibody.

Antibody fragments that recognize specific epitopes can be produced by known techniques. For example, the Fabs and Fs of the invention can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F (ab ') ₂ fragments). (ab ') ₂ fragments. The F (ab ') ₂ fragment contains a variable region, a light chain constant region, and a heavy chain CHI domain.

2. Production of anti-IL-1β monoclonal antibodies using SLAM

In another aspect of the invention, use is made of, for example, U.S. Patent No. 5,627,052; PCT Publication No. WO 92/02551; and Babcook et al., Proc. Natl. Acad. Sci. USA , 93: 7843-7848 (1996 The procedure described in), referred to in the art as the selected lymphocyte antibody method (SLAM), produces recombinant antibodies from a single, isolated lymphocyte. In this method, an antigen-specific hemolytic plaque assay is used to screen single cells that secrete relevant antibodies, such as lymphocytes derived from any of the immunized animals described in Part 1, where the antigens IL-1β, IL-1β Subunits or fragments thereof are coupled to sheep red blood cells using a linker such as biotin and are used to identify single cells that secrete antibodies specific for IL-1β. After identifying the relevant antibody-secreting cells, the heavy and light chain variable region (VH and VL) cDNAs are removed from the cells by reverse transcriptase-PCR, and then can be found in appropriate immunoglobulin constant regions (such as human constant regions). In such cases, these variable regions are expressed in mammalian host cells, such as COS or CHO cells. Further in vitro analysis and selection of host cells transfected with amplified immunoglobulin sequences derived from in vivo selected lymphocytes can be performed (e.g., by panning transfected cells) to isolate expression against IL- 1β antibody cells. The amplified immunoglobulin sequences may be further processed in vitro, such as by in vitro affinity maturation methods such as those described in PCT Publication No. WO 97/29131 and PCT Publication No. WO 00/56772.

3. Production of anti-IL-1β monoclonal antibodies using transgenic animals

In another embodiment of the invention, the antibodies are produced by immunizing a non-human animal comprising some or all human immunoglobulin loci with an IL-1β antigen. In an exemplary embodiment, the non-human animal is a XENOMOUSE transgenic mouse, which is an engineered mouse strain that contains a larger fragment of a human immunoglobulin locus and lacks mouse antibody production. See, for example, Green et al., Nature Genetics , 7: 13-21 (1994) and U.S. Patent Nos. 5,916,771; 5,939,598; 5,985,615; 5,998,209; 6,075,181; 6,091,001; 6,114,598 and 6,130,364 number. See also PCT Publication No. WO 91/10741 published on July 25, 1991; WO 94/02602 published on February 3, 1994; WO 96/34096 and WO 96/33735 published on October 31, 1996 ; WO 98/16654 published on April 23, 1998; WO 98/24893 published on June 11, 1998; WO 98/50433 published on November 12, 1998; WO 99 published on September 10, 1999 / 45031; WO 99/53049 published on October 21, 1999; WO 00/09560 published on February 24, 2000; and WO 00/037504 published on June 29, 2000. XENOMOUSE® transgenic mice produce fully human antibody-like adult human lineages and generate antigen-specific human Mabs. XENOMOUSE® transgenic mice contain approximately 80% of the human antibody lineage by introducing YAC fragments in the megabase-sized germline configuration of the human heavy chain locus and the x light chain locus. See Mendez et al., Nature Genetics 15: 146-156 (1997); and Green and Jakobovits, J. Exp. Med. 188: 483-495 (1998), the disclosures of both of which are incorporated herein by reference.

4. Production of anti-IL-1β monoclonal antibodies using recombinant antibody libraries

Antibodies of the invention can also be prepared using in vitro methods, where the antibody library is screened to identify antibodies with the specific binding specificity desired. Methods for screening recombinant antibody libraries are well known in the art and include, for example, methods described in the following literatures: Ladner et al., U.S. Patent No. 5,223,409; Kang et al., PCT Publication No. WO 92/18619; Dower et al., PCT Publication No. WO 91/17271; Winter et al., PCT Publication No. WO 92/20791; Markland et al., PCT Publication No. WO 92/15679; Breitling et al., PCT Publication No. WO 93/01288; McCarffer et al., PCT Publication No. WO 92/01047; Garrard et al., PCT Publication No. WO 92/09690; Fuchs et al., Bio / Technology , 9: 1369-1372 (1991) Hay et al., Hum . Antibod . Hybridomas , 3: 81-85 (1992); Huse et al., Science , 246: 1275-1281 (1989); McCafferty et al., Nature, 348: 552-554 (1990); Griffiths et al., EMBO J. , 12: 725-734 (1993); Hawkins et al., J. Mol. Biol. , 226: 889-896 (1992); Clackson et al., Nature , 352: 624-628 (1991 ); Gram et al., Proc. Natl. Acad. Sci. USA , 89: 3576-3580 (1992); Garrard et al., Bio / Technology , 9: 1373-1377 (1991); Hoogenboom et al., Nucl. Acids Res ., 19: 4133-4137 (1991); and Barbas et al., Proc.Natl.Acad.Sci.USA, 88: 7978-7982 (1991 ); U.S. Publication No. 2003/0186374; and PCT Publication No. WO No. 97/29131, the contents of each of which is incorporated herein by reference.

Recombinant antibody libraries can be from individuals immunized with IL-1β or a portion of IL-1β. Alternatively, the recombinant antibody library may be from an untreated individual, that is, an individual that is not immunized with IL-1β, such as a human antibody library from a human individual that is not immunized with human IL-1β. The antibodies of the invention are selected by screening a library of recombinant antibodies with a peptide comprising human IL-1β to thereby select those antibodies that recognize IL-1β. Methods for performing this screening and selection are well known in the art, such as described in the references in the previous paragraph. To select antibodies of the invention that have specific binding affinity for human IL-1β, such as antibodies that dissociate from human IL-1β at a specific K _off rate constant, surface plasmon resonance methods known in the art can be used to select K _off rate constant antibody. To select antibodies specific to the present invention having the activity of neutralizing human IL-1β, such as a specific antibody of the IC _50, standard methods may be used to inhibit the activity of human IL-1β is known in the art for evaluation.

In one aspect, the invention relates to an isolated antibody or antigen-binding portion thereof that binds human IL-1β. The antibody is preferably a neutralizing antibody. In various embodiments, the antibody is a recombinant antibody or a monoclonal antibody.

For example, various phage presentation methods known in the art can also be used to produce antibodies of the invention. In a phage presentation method, a functional antibody domain is presented on the surface of a phage particle with a polynucleotide sequence encoding it. In particular, the phage can be used to present antigen-binding domains expressed from lineages or combinatorial antibody libraries (eg, human or murine). Antigens can be used, for example, to select or identify phages that express an antigen-binding domain that binds to the relevant antigen, using labeled antigens or antigens that bind or capture on solid surfaces or beads. The phage used in these methods are usually filamentous phages, which include fd and M13 binding domains expressed by phage recombinantly fused with phage gene III or gene VIII proteins from Fab, Fv or disulfide-bonded Fv antibody domains. Examples of phage presentation methods that can be used to prepare the antibodies of the present invention include methods disclosed in the following literatures: Brinkmann et al., J. Immunol. Methods , 182: 41-50 (1995); Ames et al., J. Immunol. Methods , 184: 177-186 (1995); Kettleborough et al., Eur. J. Immunol. , 24: 952-958 (1994); Persic et al., Gene , 187: 9-18 (1997); Burton et al., Adv. Immunol. , 57: 191-280 (1994); PCT Publication No. WO 90/02809; No. WO 91/10737; No. WO 92/01047 (PCT / GB91 / 01134); No. WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Patent No. 5,698,426; No. 5,223,409; No. 5,403,484; No. 5,580,717; No. 5,427,908; No. 5,821,047; No. 5,571,698 No. 5,427,908; No. 5,516,637; No. 5,780,225; No. 5,658,727; No. 5,733,743 and No. 5,969,108; each of which is incorporated herein by reference in its entirety.

As described in the references above, after phage selection, the antibody coding region can be isolated from the phage and used to generate intact antibodies including human antibodies or any other desired antigen-binding fragments, and in any desired host, including mammalian cells , Insect cells, plant cells, yeast, and bacteria), as described in detail below, for example. For example, methods known in the art can also be used to generate recombinantly Fab, Fab ', and F (ab') 2 fragments, such as PCT Publication No. WO 92/22324; Mullinax et al., BioTechniques , 12 (6): 864-869 (1992); and Sawai et al., Am. J. Reprod. Immunol. , 34: 26-34 (1995); and Better et al., Science , 240: 1041-1043 (1988) ) (These references are incorporated by reference in their entirety). Examples of techniques that can be used to generate single chain Fv and antibodies include U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., Methods Enzymol., 203: 46-88 (1991); Shu et al., Proc. Natl. Acad. Sci . USA , 90: 7995-7999 (1993); and the technique described in Skera et al., Science , 240: 1038-1041 (1988).

Instead of screening recombinant antibody libraries by phage presentation, other methods known in the art for screening larger combinatorial libraries can be used to identify the dual specific antibodies of the invention. As described in PCT Publication No. WO 98/31700 by Szostak and Roberts and Roberts and Szostak, Proc. Natl. Acad. Sci. USA , 94: 12297-12302 (1997), a class of alternative expression systems is the expression of recombinant antibody libraries An expression system for RNA-protein fusions. In this system, a covalent fusion is formed between the mRNA and its encoded peptide or protein by translating in vitro a synthetic mRNA carrying puromycin, a peptidyl receptor antibiotic at the 3 'end. Thus, specific mRNAs can be enriched from a complex mixture of mRNAs (e.g., combinatorial libraries) based on the nature of the encoded peptide or protein (e.g., an antibody or part thereof), such as the binding of an antibody or part thereof to a dual specific antigen. Nucleic acid sequences encoding antibodies or portions thereof recovered from screening of such libraries can be expressed recombinantly as described above (e.g., in mammalian host cells), and in addition, mutant mRNA can be introduced into the originally selected sequence by pairing -Peptide fusions undergo more rounds of screening or further affinity maturation by other methods for in vitro affinity maturation of recombinant antibodies as described above.

In another method, yeast presentation methods known in the art can also be used to generate antibodies of the invention. In the yeast presentation method, a genetic method is used to tether the antibody domain to the yeast cell wall and present it on the yeast surface. In particular, the yeast can be used to present antigen-binding domains expressed from lineages or combinatorial antibody libraries (eg, human or murine). Examples of yeast presentation methods that can be used to prepare antibodies of the invention include the method disclosed by Wittrup et al. In US Patent No. 6,699,658, which is incorporated herein by reference.

B. Production of recombinant IL-1β antibodies

Antibodies of the invention can be produced by any of a number of techniques known in the art. For example, expression from a host cell, where expression vectors encoding the heavy and light chains are transfected into the host cell by standard techniques. Various forms of the term "transfection" are intended to cover Various techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-polyglucose transfection, and the like. Although it is possible to express the antibodies of the present invention in prokaryotic or eukaryotic host cells, because eukaryotic cells (and especially mammalian cells) are more likely to assemble and secrete appropriately folded and immunoactive antibodies than prokaryotic cells, It is preferred to express antibodies in these eukaryotic cells and most preferably in mammalian host cells.

Preferred mammalian host cells expressing the recombinant antibodies of the present invention include Chinese hamster ovary cells (CHO cells) (including Urlaub and Chain, Proc. Natl. Acad. Sci. USA , 77: 4216-4220 (1980)) -CHO cells for use with DHFR selectable markers, as described, for example, in Kaufman and Sharp, J. Mol. Biol., 159: 601-621 (1982)), NSO myeloma cells, COS cells, and SP2 cells . When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow the antibody to perform in the host cell or better to allow the antibody to be secreted into the medium in which the host cell is grown . Antibodies can be recovered from the culture medium using standard protein purification methods.

Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It should be understood that changes to the above procedures are within the scope of the present invention. For example, it may be desirable to transfect host cells with DNA encoding functional fragments of the light and / or heavy chains of the antibodies of the invention. Recombinant DNA technology can also be used to remove some or all of the DNA encoding one or both of the light and heavy chains that are not necessary to bind the relevant antigen. Antibodies of the invention also encompass molecules expressed from such truncated DNA molecules. In addition, bifunctional antibodies can be produced by cross-linking an antibody of the invention with a second antibody using standard chemical cross-linking methods, where one heavy chain and one light chain are antibodies of the invention and the other heavy chain and another light chain are opposite The antigen of the relevant antigen is specific.

In an exemplary system that recombinantly expresses an antibody or antigen-binding portion thereof of the present invention, a recombinant expression vector encoding an antibody heavy chain and an antibody light chain is introduced by calcium phosphate-mediated transfection dhfr-CHO cells. Within the recombinant expression vector, the antibody heavy chain and light chain genes are each operably linked to CMV enhancer / AdMLP promoter regulatory elements to drive gene transcription at a high level. The recombinant expression vector also carries the DHFR gene, which allows the use of methotrexate selection / amplification to select CHO cells that have been transfected with the vector. The selected transformant host cells are cultured to allow expression of antibody heavy and light chains and recovery of intact antibodies from the culture medium. Recombinant expression vectors are prepared using standard molecular biology techniques, host cells are transfected, transformants are selected, host cells are cultured, and antibodies are recovered from the culture medium. In addition, the present invention provides a method for synthesizing the recombinant antibody of the present invention by culturing the host cell of the present invention in a suitable medium until the recombinant antibody of the present invention is synthesized. The method may further include isolating the recombinant antibody from the culture medium.

Anti-human IL-1β chimeric antibody

Chimeric antibodies are molecules from which different parts of the antibody are derived from different animal species, such as antibodies with variable regions derived from murine monoclonal antibodies and human immunoglobulin constant regions. Methods of generating chimeric antibodies are known in the art and are discussed in detail in the Examples section. See, eg, Morrison, SL, Science , 229: 1202-1207 (1985); Oi et al., BioTechniques , 4: 214-221 (1986); Gillies et al., J. Immunol. Methods , 125: 191-220 (1989) US Patent Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entirety. In addition, techniques developed to generate "chimeric antibodies" by splicing genes from mouse antibody molecules with appropriate antigen specificity and genes from human antibody molecules with appropriate biological activity can be used (Morrison Et al., Proc. Natl. Acad. Sci. USA , 81: 6851-6855 (1984); Neuberger et al., Nature , 312: 604-608 (1984); Takeda et al., Nature , 314: 452-454 (1985) ), Which is incorporated herein by reference in its entirety).

In one example, the chimeric anti-system of the present invention is prepared by replacing the heavy chain constant region of the murine single anti-human IL-1β antibody described in Section 1 with the human IgG1 constant region.

2. Anti-IL-1β CDR grafted antibody

CDR-grafted antibody of the present invention comprises a heavy and light chain variable region sequences from a human antibody in which V _H and / or one or more CDR regions V _L CDR sequences of murine antibodies by the present invention substitutions. Framework sequences from any human antibody can serve as a template for CDR transplantation. However, direct chain replacement on such frameworks usually results in some loss of binding affinity to the antigen. The higher the homology between the human antibody and the original murine antibody, the less likely it is that the combination of the murine CDR and the human framework will introduce aberrations that reduce affinity. Therefore, human variable frameworks selected to replace murine variable frameworks other than CDRs preferably have at least 65% sequence identity with murine antibody variable region frameworks. Human and murine variable regions other than CDRs preferably have at least 70% sequence identity. Human and murine variable regions other than CDRs even have at least 75% sequence identity. Human and murine variable regions other than CDRs preferably have at least 80% sequence identity. Methods for generating chimeric antibodies are known in the art. See, for example, European Patent No. EP 0 239 400; PCT Publication No. WO 91/09967; US Patent No. 5,225,539; No. 5,530,101; and No. 5,585,089. For the decoration or remodeling of antibodies, see, for example, European Patent Nos. EP 0 592 106 and EP 0 519 596; Padlan, Mol. Immunol. , 28 (4/5): 489-498 (1991); Studnicka et al. Human, Protein Eng., 7 (6): 805-814 (1994); and Roguska et al., Proc. Natl. Acad. Sci. USA , 91: 969-973 (1994). For antibody chain shuffling, see, for example, US Patent No. 5,565,352.

3. Anti-human IL-1β humanized antibody

A humanized antibody is an antibody molecule derived from a non-human species antibody that binds a desired antigen and has one or more complementarity determining regions (CDRs) from a non-human species antibody and a framework region from a human immunoglobulin molecule. Known human Ig sequences are disclosed, for example, at the following global information website: www.ncbi.nlm.nih.gov/entrez-/query.fcgi; www.atcc.org/phage/hdb.html; www.sciquest.com/; www .abcam.com /; www.antibodyresource.com/onlinecomp.html; www.public.iastate.edu/.about.pedro/research_tools.html; www.mgen.uni-heidelberg.de/SD/IT/IT.html; www.whfreeman.com/immunology/CH-05/kuby05.htm; www.library.thinkquest.org/12429/Immune/Antibody.html; www.hhmi.org/grants/lectures/1996/vlab/; www.path.cam.ac.uk/.about.mrc7/m-ikeimages.html; www.antibodyresource.com/; mcb.harvard.edu/BioLinks /Immuno-logy.html.www.immunologylink.com/; pathbox.wustl.edu/.about.hcenter/index.-html; www.biotech.ufl.edu/.about.hcl/; www.pebio.com/ pa / 340913 / 340913.html-; www.nal.usda.gov/awic/pubs/antibody/; www.m.ehime-u.acjp / .about.yasuhito- / Elisa.html; www.biodesign.com/ table.asp; www.icnet.uk/axp/facs/davies/lin-ks.html; www.biotech.ufl.edu/.about.fccl/protocol.html; www.isac-net.org/sites_geo.html ; Aximtl.imt.uni-marburg.de/.about.rek/AEP-Start.html; baserv.uci.kun.nl/.about.jraats/linksl.html; www.recab.uni-hd.de/immuno .bme.nwu.edu /; www.mrc-cpe.cam.ac.uk/imt-doc/pu-blic/INTRO.html; www.ibt.unam.mx/vir/V_mice.html; imgt.cnusc. fr: 8104 /; www.biochem.ucl.ac.uk/.about.martin/abs/index.html antibody.bath.ac.uk/; abgen.cvm.tamu.edu/lab/wwwabgen.html; www.unizh.ch/.about.honegger/AHOsem-inar/Slide01.html; www.cryst.bbk.ac. uk / .about.ubcg07s /; www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm; www.path.cam.ac.uk/.about.mrc7/h-umanisation/TAHHP.html; www .ibt.unam.mx / vir / structure / stat_aim.html; www.biosci.missouri.edu/smithgp/index.html; www.cryst.bioc.cam.ac.uk/.abo-ut.fmolina/Web- pages / Pept / spottech.html; www.jerini.de/fr roducts.htm; www.patents.ibm.com/ibm.html; Kabat et al., Sequences of Proteins of Immunological Interest, USDept.Health (1983), each of which is incorporated by reference in its entirety and Included in this article. Such input sequences can be used to reduce immunogenicity, or to reduce, enhance, or alter binding, affinity, rate of association, rate of dissociation, avidity, specificity, half-life, or any other known in the art Suitable for characteristics.

Framework (FR) residues in the human framework region can be substituted with corresponding residues from a CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, such as by modeling interactions between CDRs and framework residues to identify framework residues important for antigen binding and performing sequence comparisons to identify specific positions. Abnormal framework residues. See, eg, Queen et al., U.S. Patent No. 5,585,089; Riechmann et al., Nature 332: 323-327 (1988), which are incorporated herein by reference in their entirety. Three-dimensional immunoglobulin models are commonly available and are well known to those skilled in the art. Computer programs are available that illustrate and present probable three-dimensional conformations of selected candidate immunoglobulin sequences. A review of these presentations allowed analysis of the role that residues may play in the functioning of candidate immunoglobulin sequences, that is, residues that affect the ability of a candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from common and input sequences to achieve desired antibody characteristics, such as increased affinity for the target antigen. In general, CDR residues are directly and most substantially involved in affecting antigen binding. Antibodies can be humanized using a variety of techniques known in the art, such as, but not limited to, those described in the following documents: Jones et al., Nature, 321: 522-525 (1986); Verhoeyen et al., Science, 239: 1534-1536 (1988); Sims et al. , J. Immunol., 151: 2296-2308 (1993); Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987); Carter Et al., Proc. Natl. Acad. Sci. USA, 89: 4285-4289 (1992); Presta et al. , J. Immunol. , 151: 2623-2632 (1993); Padlan, Mol. Immunol., 28 (4 / 5): 489-498 (1991); Studnicka et al., Protein Eng. , 7 (6): 805-814 (1994); Roguska et al., Proc. Natl. Acad. Sci. USA, 91: 969-973 (1994); PCT Publication No. WO 91/09967; No. WO 90/14443; No. WO 90/14424; No. WO 90/14430; No. WO 99/06834 (PCT / US98 / 16280); No. WO 97/20032 (PCT / US96 / 18978); WO 92/11272 (PCT / US91 / 09630); WO 92/03461 (PCT / US91 / 05939); WO 94/18219 (PCT / US94 / 01234); WO 92/01047 (PCT / GB91 / 01134); and WO 93/06213 (PCT / GB92 / 01755); European Patent No. EP 0 592 106; No. EP 0 519 596 No. and EP 0 239 400; U.S. Patent Nos. 5,565,332; 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352 No. 6,204,023; No. 6,180,370; No. 5,693,762; No. 5,530,101; No. 5,585,089; No. 5,225,539 and No. 4,816,567, each of which is incorporated herein by reference in its entirety, including the references cited therein.

5. Anti-IL-1β DVD-Ig ^TM Binding protein

Dual variable domain immunoglobulin binding proteins (DVD-Ig) that bind to one or more epitopes of IL-1β are also provided. The DVD-Ig binding protein can also bind to the epitope of IL-1β and the epitope of a second target antigen other than the IL-1β polypeptide. An exemplary embodiment of these DVD-Ig molecules includes: a heavy chain including the structural formula VD1- (X1) n-VD2-C- (X2) n, where VD1 is the first heavy chain variable domain and VD2 is the first The variable domain of the double chain, C is the constant domain of the heavy chain, X1 is the linker, with the limitation that it is not CH1, X2 is the Fc region, and n is 0 or 1, but preferably 1; and contains the structural formula VD1- (X1) n-VD2-C- (X2) n light chain, where VD1 is the first light chain variable domain, VD2 is the second light chain variable domain, C is the light chain constant domain, and X1 is the linker, The limiting conditions are that it is not CH1, and X2 does not contain an Fc region; and n is 0 or 1, and preferably 1. The DVD-Ig may include two such heavy chains and two such light chains, wherein each chain includes variable domains connected in series, and there is no constant region inserted between the variable regions. The middle heavy chain and the light chain associate to form two tandem antigen binding sites, and one pair of heavy and light chains can associate with another pair of heavy and light chains to form a tetramer with four antigen binding sites Binding protein. In another embodiment, the DVD-Ig molecule may include a heavy chain and a light chain each including three variable domains (eg, VD1, VD2, VD3) connected in series, with no constant regions inserted between the variable domains, One pair of heavy and light chains can associate to form three antigen-binding sites, and one pair of heavy and light chains can associate with another pair of heavy and light chains to form four with six antigen-binding sites Polymer-binding protein.

Each variable domain (VD) in DVD-Ig can be obtained from one or more "parents" that bind one or more desired antigens or epitopes, such as IL-1β and / or IL-1α antigens or epitopes. Monoclonal antibodies.

A. Generation of parental monoclonal antibodies

The variable domain of a DVD-Ig binding protein can be obtained from a parent antibody (including a monoclonal antibody (mAb)) capable of binding the relevant antigen. These antibodies can be produced naturally or by recombinant techniques. It should be understood that if the antibody that binds the desired target antigen or epitope is a multiple strain antibody, it is still necessary to obtain the antigen-binding site of a single antibody from a multiple strain population (that is, a single single member of the multiple strain population). Variable domain for DVD-Ig generation. Monoclonal antibodies can be produced by any of a variety of methods known in the art, including those described herein (see sections A.1.-A.4. Above).

B. Criteria for selecting parental monoclonal antibodies

One embodiment of the present invention relates to the selection of a parent antibody having at least one or more of the properties required in a DVD-Ig molecule. In one embodiment, the desired property is selected from one or more antibody parameters. In another embodiment, the antibody parameter is selected from the group consisting of: antigen specificity, affinity with the antigen, potency, biological function, epitope recognition, stability, solubility, production efficiency, immunogenicity, drug Kinetics, bioavailability, tissue cross-reactivity and orthologous antigen binding.

B1. Affinity to antigen

The required affinity of a therapeutic mAb may depend on the nature of the antigen and the desired treatment endpoint. In one embodiment, because the cytokine-cytokine receptor interaction is usually a high-affinity interaction (e.g., <pM- <nM range), when the monoclonal antibody blocks the interaction, the monoclonal antibody has a higher Affinity (Kd = 0.01-0.50pM). In such cases, the affinity of the mAb for its target should equal or exceed the affinity of the cytokine (ligand) for its receptor. On the other hand, mAbs with smaller affinities (> nM range) can be therapeutically effective, for example, in clearing circulating potentially pathogenic proteins, such as binding, chelating, and removing target antigens such as A-β amyloid Monoclonal antibodies against circulating substances. In other cases, site-directed mutagenesis can be used to reduce the affinity of an existing high-affinity mAb or use a mAb that has a lower affinity for its target can be used to avoid potential side effects, such as a high-affinity mAb can chelate or neutralize all of its intended targets Target, thereby completely depleting / eliminating the function of the targeted protein. In this case, the low-affinity mAb can chelate / neutralize a portion of the target (pathological or excessively produced) that may cause disease symptoms, thus allowing a portion of the target to continue to perform its normal physiological function. Therefore, Kd may be reduced to adjust the dose and / or reduce side effects. The affinity of the parental mAb can play a role in properly targeting cell surface molecules to achieve the desired therapeutic result. For example, if the target is expressed on cancer cells at high density and on normal cells at low density, lower affinity mAb will bind a larger number of targets on tumor cells than normal cells, resulting in Tumor cells are eliminated via ADCC or CDC, and thus may have the desired therapeutic effect. Therefore, selecting a mAb with the desired affinity can be relevant for both soluble and surface targets.

Signaling after the receptor interacts with its ligand depends on the affinity of the receptor-ligand interaction. Similarly, it is envisaged that the affinity of the mAb for surface receptors can determine the nature of intracellular signaling and whether the mAb can transmit a potent signal or an antagonist signal. The affinity-based nature of mAb-mediated signaling can affect its profile of side effects. Therefore, the required affinity and required function of a therapeutic monoclonal antibody must be carefully determined by in vitro and in vivo experiments.

The required Kd of a binding protein (eg, an antibody) can be determined experimentally based on the desired treatment result. In one embodiment, a parent antibody is selected that has an affinity (Kd) for a particular antigen that is equal to or exceeds the required affinity of DVD-Ig for the same antigen. Antigen binding affinity and kinetics were evaluated by Biacore or another similar technique. In one embodiment, each parent antibody has a dissociation constant (Kd) with its antigen selected from the group consisting of: at most about 10 ^-7 M, at most about 10 ^-8 M, at most about 10 ^-9 M, at most about 10 ^-10 M, at most about 10 ^-11 M, at most about 10 ^-12 M, and at most 10 ^-13 M. The first parental antibody from which VD1 is obtained and the second parental antibody from which VD2 is obtained may have similar or different affinities (K _D ) to the corresponding antigen. Each parent antibody has an association rate constant (Kon) with an antigen selected from the group consisting of: at least about 10 ² M ^-1 s ^-1 , at least about 10 ³ as measured by surface plasmon resonance. M ^-1 s ^-1 , at least about 10 ⁴ M ^-1 s ^-1 , at least about 10 ⁵ M ^-1 s ^-1 , and at least about 10 ⁶ M ^-1 s ^-1 . The first parental antibody from which, for example, VD1 is obtained, and the second parental antibody from which VD2 is obtained, may have similar or different association rate constants (Kon) for the corresponding antigen. In one embodiment, each parent antibody has a dissociation rate constant (Koff) from an antigen selected from the group consisting of: at most about 10 ^-3 s ^-1 , at most, as measured by surface plasmon resonance. About 10 ^-4 s ^-1 , at most about 10 ^-5 s ^-1 , and at most about 10 ^-6 s ^-1 . The first parental antibody from which VD1 is obtained and the second parental antibody from which VD2 is obtained may have similar or different off-rate constants (Koff) for the corresponding antigen.

B2. Effectiveness

The required affinity / potency of the parental monoclonal antibody depends on the desired treatment outcome. For example, for receptor-ligand (R-L) interactions, the affinity (kd) is equal to or exceeds R-L kd (pM range). For simple clearance of pathological circulating proteins, Kd can be in the low nM range, such as clearing various substances of circulating A-β peptides. In addition, Kd also depends on whether the target exhibits multiple copies of the same epitope, such as mAb that targets conformational epitopes in Aβ oligomers.

Where VDI and VD2 bind the same antigen but bind different epitopes, DVD-Ig contains binding sites for the same antigen, thus increasing the affinity and thus the apparent Kd of DVD-Ig. In one embodiment, a parent antibody is selected that has a kd that is equal to or lower than the desired Kd in DVD-Ig. The affinity consideration of the parental mAb may also depend on whether the DVD-Ig contains four or more identical antigen binding sites (ie, the DVD-Ig is from a single mAb). In this case, the apparent kd will be greater than the mAb due to the affinity. These DVD-Igs can be used to cross-link surface receptors, increase neutralization efficacy, and enhance the clearance of pathological proteins.

In another embodiment, a parent antibody is selected that has a neutralizing potency against specific antigens that is equal to or exceeds the required neutralization potential of DVD-Ig for the same antigen. Neutralizing efficacy can be assessed by target-dependent bioassays, where appropriate types of cells generate measurable signals (i.e., proliferation or cytokine production) in response to target stimulation, and are neutralized by targets of mAb Signaling can be reduced in a dose-dependent manner.

B3. Biological functions

Monoclonal antibodies can potentially perform several functions. Some of these functions are listed in Table 5. These functions can be evaluated by in vitro assays (such as cell-based assays and biochemical assays) and in vivo animal models.

MAbs with different functions described in the examples herein and in Table 5 can be selected to achieve the desired therapeutic results. Two or more selected parental monoclonal antibodies can then be used as DVD-Ig The form is used to accomplish two different functions in a single DVD-Ig molecule. For example, DVD-Ig can be generated by selecting a parental mAb that neutralizes the function of a particular cytokine (such as IL-1β) and selecting a parental mAb that enhances the clearance of pathological proteins. Similarly, two parental mAbs can be selected that recognize two different cell surface receptors, one mAb having a potentiating function against one receptor and the other mAb having an antagonistic function against another receptor. These two selected mAbs, each with different functions, can be used to construct a single DVD-Ig molecule with two different functions (agonists and antagonists) of the selected monoclonal antibody in a single molecule. Similarly, two antagonistic mAbs, each of which is a cell surface receptor that blocks the binding of the corresponding receptor ligands (eg, EGF and IGF), can be used in the form of DVD-Ig. Conversely, an antagonistic anti-receptor mAb (e.g., anti-EGFR) and a neutralizing anti-soluble mediator (e.g., anti-IGF1 / 2) mAb can be selected to make DVD-Ig.

B4. Epitope recognition:

Different regions of the protein can perform different functions. For example, specific regions of a cytokine (such as IL-1β) interact with cytokine receptors to cause receptor activation, while other regions of the protein may be required to stabilize cytokines. In this case, a mAb that specifically binds to a receptor interaction region on a cytokine and thereby blocks the cytokine-receptor interaction can be selected. In some cases, for example, in the case of certain chemokine receptors that bind to multiple ligands, they may choose to bind to an epitope (region on the chemokine receptor) that interacts with only one ligand MAb. In other cases, monoclonal antibodies can bind to epitopes on the target that are not directly responsible for the physiological function of the protein, but the binding of the mAb to these regions can interfere with the physiological function (steric hindrance) of the protein or change the configuration of the protein So that the protein cannot function (the mAb of a receptor with multiple ligands changes the configuration of the receptor so that no one ligand can bind). Anti-cytokine monoclonal antibodies (eg, 125-2H, an anti-IL-18 mAb) that do not block the binding of cytokines to their receptors but block signal transduction have also been identified.

Examples of epitope and mAb functions include, but are not limited to, blocking receptor-ligand (R-L) interactions (binding to neutralizing mAb at R interaction sites); resulting in reduced R binding Or without steric hindrance of R binding. Antibodies can bind to targets at sites other than the receptor binding site, but still induce conformational changes and eliminate functions (e.g. XOLAIR® omalizumab, Genetech / Novartis), bind to R but Blocks signalling (125-2H mAb) and interferes with receptor binding and target function.

In one embodiment, the parental mAb needs to target the appropriate epitope for maximum efficacy. This epitope should be conserved in DVD-Ig. The binding epitope of mAb can be determined by several methods, including co-crystallization, limited proteolysis of mAb-antigen complexes, and peptide localization by mass spectrometry (Legros et al., Protein Sci., 9: 1002-1010 (2000) ), Phage-presenting peptide library (O'Connor et al. , J. Immunol. Methods., 299: 21-35 (2005)) and mutation induction (Wu C. et al. , J. Immunol. , 170: 5571-5577 ( 2003)).

B5. Physical Chemistry and Pharmaceutical Properties

Therapeutic treatment with antibodies often requires the administration of high doses, usually a few mg / kg (due to the low potency by mass due to the usually large molecular weight). In order to adapt to patient compliance and fully invest in long-term disease therapy and outpatient treatment, subcutaneous (s.c.) or intramuscular (i.m.) administration of therapeutic mAb is desirable. For example, the maximum required volume for subcutaneous administration is about 1.0 mL, and therefore a concentration of> 100 mg / mL is needed to limit the number of injections per dose. In one embodiment, the therapeutic antibody is administered in a single dose. However, the development of these formulations is constrained by protein-protein interactions (e.g., aggregation that potentially increases the risk of immunogenicity) and by constraints (e.g., viscosity) during processing and delivery. Therefore, the large number of clinical efficacy and related development constraints limit the potential for antibody formulations and the full utilization of subcutaneous administration in high-dose regimens. Obviously, the physicochemical and pharmaceutical properties of protein molecules and protein solutions are very important, such as stability, solubility and viscosity characteristics.

B5.1. Stability

A `` stable '' antibody formulation is one in which the antibody essentially retains its physical stability after storage Qualitative and / or chemically stable and / or biologically active formulations. Stability can be measured at a selected temperature for a selected period of time. In one embodiment, the antibody in the formulation is stable at room temperature (about 30 ° C) or at 40 ° C for at least 1 month and / or at about 2-8 ° C for at least 1 year, such as at least 2 years. Furthermore, in one embodiment, the formulation is stable after the formulation is frozen (to, for example, -70 ° C) and thawed (hereinafter referred to as a "freeze / thaw cycle"). In another example, a "stable" formulation may be a formulation in which less than about 10% and less than about 5% of the protein is present in the formulation as an aggregate.

DVD-Ig that is stable in vitro at various temperatures for extended periods of time is desirable. This can be achieved by rapid screening of parental mAbs that are stable in vitro at elevated temperatures (e.g., 40 ° C) for 2-4 weeks and then assessing stability. During storage at 2-8 ° C, the protein shows stability for at least 12 months, such as at least 24 months. Various techniques can be used to assess stability (monomer,% of intact molecules), such as cation exchange chromatography, size exclusion chromatography, SDS-PAGE, and bioactivity testing. For a more comprehensive list of analytical techniques that can be used to analyze covalent and conformational modifications, see Jones, AJS, "Analytical methods for the assessment of protein formulations and delivery systems", Chapter 2, Formulation and delivery of peptides and proteins , Section 1st edition, (ed. Cleland and Langer) (American Chemical Society, Washington, DC, 1994) pages 22-45; and Pearlman and Nguyen, "Analysis of Protein drugs", Chapter 6, Peptide and protein drug delivery , 1 Edition [Advances in Parenteral Sciences, Volume 4] (Lee, VH) (Marcel Dekker, Inc., New York, 1991) pp. 247-301.

Heterogeneity and aggregate formation: The stability of the antibody can allow the formulation to show less than about 10%, and in one embodiment less than about 5%, in another embodiment less than about 2%, or in In one embodiment, the GMP antibody substance exists in the form of an aggregate in a range of 0.5% to 1.5% or less. Size exclusion chromatography is a sensitive, reproducible, and extremely stable method for detecting protein aggregates.

In addition to low aggregate content, antibodies must be chemically stable in one embodiment. Chemical stability can be determined by ion exchange chromatography (such as cation or anion exchange chromatography), hydrophobic interaction chromatography, or other methods (such as isoelectric focusing or capillary electrophoresis). For example, the chemical stability of antibodies can increase the peak value of representative unmodified antibodies in cation exchange chromatography after storage at 2-8 ° C for at least 12 months compared to the antibody solution before the storage test. Not more than 20%, not more than 10% in one embodiment, or not more than 5% in another embodiment.

In one embodiment, the parent antibody displays structural integrity; proper disulfide bond formation, and proper folding: chemical instability due to changes in the secondary or tertiary structure of the antibody can affect antibody activity. For example, the stability indicated by the activity of the antibody can be such that after storage at 2-8 ° C for at least 12 months, the activity of the antibody can be reduced by no more than 50% compared to the antibody solution before the storage test. No more than 30%, or even no more than 10%, or no more than 5% or 1% in one embodiment. Suitable antigen binding assays can be used to determine antibody activity.

B5.2. Solubility

The "solubility" of mAb is related to the production of properly folded monomeric IgG. So the solubility of IgG can be evaluated by HPLC. For example, soluble (monomeric) IgG will generate a single peak on the HPLC chromatogram, while insoluble (e.g., multimerization and aggregation) IgG will generate multiple peaks. Those skilled in the art will therefore be able to detect increases or decreases in the solubility of IgG using conventional HPLC techniques. For a more comprehensive list of analytical techniques that can be used to analyze solubility, see Jones, AG, Dep. Chem. Biochem. Eng., Univ. Coll. London, "Particle formation and separation in suspension crystallization processes", Chapter 4, Process. Solid-Liquid Suspensions, (edited by P. Ayazi Shamlou) (Butterworth-Heinemann, Oxford, UK, 1993) pp. 93-117; and Pearlman and Nguyen, "Analysis of protein drugs", Chapter 6, Peptide and protein drug delivery , 1st edition [Advances in Parenteral Sciences, Volume 4] (Lee, VH) (Marcel Dekker, Inc., New York, 1991) pp. 247-301. The solubility of a therapeutic mAb is critical to formulating the high concentration that is often required for adequate administration. As outlined herein, a solubility of greater than 100 mg / mL may be required to provide effective antibody administration. For example, the solubility of the antibody may be not less than about 5 mg / mL in the early research stage, in one embodiment, not less than about 25 mg / mL in the advanced process science stage, or in one embodiment, not less than about 100 mg / mL. mL, or in one embodiment, may not be less than about 150 mg / mL. The intrinsic properties of protein molecules are important to the physicochemical properties (eg, stability, solubility, viscosity) of the protein solution. However, those skilled in the art will appreciate that there are a variety of excipients that can be used as additives to favorably affect the characteristics of the final protein formulation. Such excipients may include: (i) liquid solvents, co-solvents (e.g. alcohols such as ethanol); (ii) buffering agents (e.g. phosphate, acetate, citrate, amino acid buffers); ( iii) sugar or sugar alcohols (e.g. sucrose, trehalose, fructose, raffinose, mannitol, sorbitol, polydextrose); (iv) surfactants (e.g. polysorbate 20, polysorbate 40 , Polysorbate 60, polysorbate 80, poloxamer); (v) isotonicity modifiers (e.g., salts such as NaCl, sugars, sugar alcohols); and (vi) other additives Excipients (e.g. preservatives, chelating agents, antioxidants, chelating substances (e.g. EDTA), biodegradable polymers, carrier molecules (e.g. HSA, PEG).

Viscosity is a very important parameter for antibody manufacturing and antibody processing (e.g. diafiltration / ultrafiltration), filling-finishing process (pumping mode, filtering mode), and delivery mode (injectability, complex device delivery) . Low viscosity makes liquid solutions with higher concentrations of antibodies possible. This allows the same dose to be administered in a smaller volume. Smaller injection volumes have the advantage of lower pain during injection, and the solution does not necessarily need to be isotonic to reduce pain during injection. The viscosity of the antibody solution can be such that the viscosity of the antibody solution is less than 200 mPa s at a shear rate of 100 (1 / s), less than 125 mPa s in one embodiment, and less than 70 mPa s in another embodiment, and In another embodiment it is below 25 mPa s or even below 10 mPa s.

B5.3. Production efficiency

The production of DVD-Ig that is effectively expressed in mammalian cells, such as Chinese Hamster Ovary Cells (CHO), requires, in one embodiment, two parental monoclonal antibodies that are themselves effective in mammalian cells. The productivity of a self-stabilizing mammalian strain (i.e., CHO) should be above about 0.5 g / L, in one embodiment above about 1 g / L, and in another embodiment between about 2 g / L and about 5 g / L L or about 5 g / L or more (Kipriyanov et al . , Mol . Biotechnol. , 12: 173-201 (1999); Carroll et al., Expert Opin Biol Ther. , 4: 1821-1829 (2004)).

The production of antibodies and Ig fusion proteins in mammalian cells is affected by several factors. Engineered expression vectors by incorporating strong promoters, enhancers and selectable markers can maximize the transcription of related genes from an integrated vector copy. Identification of vector integration sites that allow high-level gene transcription can enhance protein expression from the vector (Wurm, FM, Nature Biotechnol. , 22 (11): 1393-1398 (2004)). In addition, the amount of production is affected by the ratio of antibody heavy chains to light chains and various steps in the process of protein assembly and secretion (Jiang et al., Biotechnol. Prog. , 22 (1): 313-318 (2006)).

B6. Immunogenicity

The administration of a therapeutic mAb can lead to a specific occurrence of an immune response (ie, the formation of endogenous antibodies against the therapeutic mAb). Potential elements that can induce immunogenicity should be analyzed during selection of parental monoclonal antibodies, and steps to reduce this risk can be taken to optimize parental monoclonal antibodies before DVD-Ig construction. Mouse-derived antibodies have been found to be highly immunogenic in patients. The next step in the generation of chimeric antibodies comprising mouse variable regions and human constant regions presents a reduction in the immunogenicity of therapeutic antibodies (Morrison and Schlom, "Recombinant Chimeric Monoclonal Antibodies", Chapter 1, Important Advances in Oncology 1990 (JBLippincott Company, Philadelphia, 1990) pp. 3-18). Alternatively, as described by Riechmann et al., Nature , 332: 323-327 (1988) on therapeutic antibodies, the murine CDR sequences can be transferred into the human antibody framework (reshaping / CDR grafting / human ) To reduce immunogenicity. Another method is called "surface remodeling" or "decoration", starting with rodent variable light and heavy chain domains, and changing only surface accessible framework amino acids into human framework amino acids The CDRs and embedded amino acids are still derived from the parent rodent antibody (Roguska et al., Protein Eng. , 9 (10): 895-904 (1996)). In another type of humanization, instead of transplanting the entire CDR, one technique transplants only the "specificity determining region" (SDR), which is defined as a subset of CDR residues involved in the binding of an antibody to its target (Kashmiri et al., Methods, 36 (1): 25-34 (2005)). This requires identifying SDRs by analyzing the available three-dimensional structure of the antibody-target complex or performing mutation analysis on the antibody CDR residues to determine which interacts with the target. Alternatively, fully human antibodies may have reduced immunogenicity compared to murine, chimeric, or humanized antibodies.

Another way to reduce the immunogenicity of therapeutic antibodies is to eliminate certain sequences that are predicted to be immunogenic. In one approach, after the first-generation biologics have been tested in humans and found to have unacceptable immunogenicity, B-cell epitopes can be localized and subsequently altered to avoid immune detection. Another method uses a method that predicts and removes potential T-cell epitopes. Computational methods have been developed to scan and identify peptide sequences of biotherapeutic agents with the potential to bind MHC proteins (Desmet et al., Proteins, 58: 53-69 (2005)). Alternatively, human dendritic cell-based methods can be used to identify CD4 ⁺ T cell epitopes in potential protein allergens (Stickler et al., J. Immunother., 23: 654-660 (2000); Morrison and Schlom, Important Adv. Oncol. (1990) pp. 3-18; Riechmann et al., "Reshaping human antibodies for therapy", Nature. 332: 323-327 (1988); Roguska et al., "A comparison of two murine mAbs humanized by CDR -grafting and variable domain resurfacing ", Protein Eng. , 9: 895-904 (1996); Kashmiri et al.," SDR grafting--a new approach to antibody humanization ", Methods, 36 (1): 25-34 (2005 ); Desmet et al., "Anchor profiles of HLA-specific peptides: analysis by a novel affinity scoring method and experimental validation", Proteins, 58: 53-69 (2005); Stickler et al., "CD4 + T-cell epitope determination using unexposed human donor peripheral blood mononuclear cells ", J. Immunother., 23: 654-660 (2000)).

B7. In vivo efficacy

In order to generate a DVD-Ig molecule with the desired in vivo efficacy, when presented in combination, its importance is to generate and select a mAb with similar desired in vivo efficacy. However, in some cases, DVD-Ig can exhibit in vivo efficacy that cannot be achieved by a combination of two independent mAbs. For example, DVD-Ig can bring two targets in close proximity, resulting in an activity that cannot be achieved by a combination of two independent mAbs. Other desirable biological functions are described herein in Section B3 . Selection of the characteristics required for a DVD-Ig molecule can be based on factors such as pharmacokinetic half-life (t½), tissue distribution, soluble amount relative to the cell surface target, and target concentration-soluble amount / density-surface Parent antibody.

B8. Tissue distribution in vivo

In order to generate a DVD-Ig molecule with a desired in vivo tissue distribution, in one embodiment, a parent mAb with a similar desired in vivo tissue distribution profile must be selected. Alternatively, based on a mechanism based on a dual specific targeting strategy, at other times when presented in combination, it may not be necessary to select a parent mAb with a similar desired tissue distribution in vivo. For example, in the case of DVD-Ig, one of the binding components targets the DVD-Ig to a specific site, thereby directing the second binding component to the same target site. For example, one of the binding specificities of DVD-Ig can target the pancreas (islet cells) and the other specificity can direct GLP1 to the pancreas to induce insulin.

B9. Isotype

In order to generate a DVD-Ig molecule having desired properties including, but not limited to, isotype, effector function, and circulatory half-life, a parent mAb with appropriate Fc effector function is selected depending on the therapeutic utility and the desired therapeutic endpoint. There are five major heavy chain species or isotypes (some of which have several subtypes) and these determine the effector function of the antibody molecule. These effector functions exist in anti- Body molecules in the hinge region, CH2 and CH3 domains. However, residues in other parts of the antibody molecule can also affect effector functions. Hinge region Fc effector functions include: (i) antibody-dependent cytotoxicity (ADCC), (ii) complement (C1q) binding, activation and complement-dependent cytotoxicity (CDC), and (iii) phagocytosis of antigen-antibody complexes / Clearance and (iv) cytokine release in some cases. These Fc effector functions of antibody molecules are mediated through the interaction of the Fc region with a set of species-specific cell surface receptors. IgG1 isotype antibodies are the most active, while IgG2 and IgG4 have minimal or no effect. The effector functions of IgG antibodies are mediated through interactions with three structurally homologous cellular Fc receptor types (and subtypes) (FcgR1, FcgRII, and FcgRIII). These effector functions of IgG1 can be eliminated by mutating specific amino acid residues (such as L234A, L235A) in the lower hinge region required for FcgR and C1q binding. The amino acid residues in the Fc region, especially in the CH2-CH3 domain, also determine the circulating half-life of the antibody molecule. This Fc function is mediated by the binding of the Fc region to the neonatal Fc receptor (FcRn), which is responsible for the recycling of antibody molecules from acid lysosomes back to the whole body cycle.

Whether an mAb is active or inactive depends on the desired therapeutic endpoint of the antibody. Some examples of the use of isoforms and desired therapeutic results are listed below: 1. If the desired endpoint is the functional neutralization of soluble cytokines, an inactive isoform can be used; 2. If the desired result is the removal of pathological proteins, then Use active isoforms; 3. Use active isoforms if desired result is clearance of protein aggregates; 4. Use inactive isoforms if desired result is antagonistic to surface receptors (Tysabri, IgG4; OKT3®, mutant IgG1) ; 5. If the desired result is elimination of target cells, use active isotypes (Herceptin, IgG1 (and have enhanced effector functions)); and 6. If the desired result is to clear proteins from circulation without entering the CNS, then Use IgM isotypes (e.g., to remove circulating Ab peptide material).

The Fc effector function of the parental mAb can be controlled by various in vitro methods well known in the art. Method determination.

As discussed, the choice of isotype, and thus the effector function, will depend on the desired end point of treatment. In the case where it is only necessary to neutralize circulating targets, such as blocking receptor-ligand interactions, effector functions may not be needed. In such cases, mutations in the isotype or antibody Fc region of the effector function need to be eliminated. In other cases where the elimination of target cells is a therapeutic endpoint (e.g., tumor cell elimination), mutations or defucosylation in the isotype or Fc region that enhance effector function are required (Presta, LG, Adv. Drug Del. Rev. 58: 640-656 (2006); Satoh et al. Expert Opin. Biol. Ther. , 6: 1161-1173 (2006)). Similarly, depending on the therapeutic effect, the circulating half-life of antibody molecules can be shortened / prolonged by introducing specific mutations in the Fc region to modulate antibody-FcRn interactions (Dall'Acqua et al., J. Biol. Chem. , 281 : 23514-23524 (2006); Petkova et al., Int. Immunol. , 18: 1759-1769 (2006); Vaccaro et al., Proc. Natl. Acad. Sci. USA , 103: 18709-18714 (2006)).

Public information on the various residues affecting the different effector functions of normal therapeutic mAbs may require confirmation of DVD-Ig. In the DVD-Ig format, other (different) Fc region residues than the residues identified for modulating the effector function of the monoclonal antibody may be important.

Overall, the decision as to what Fc effector function (isotype) is crucial in the final DVD-Ig format depends on the disease indication, the target of treatment, the desired end point of treatment, and safety considerations. Exemplary suitable heavy and light chain constant regions are listed below, including (but not limited to): IgG1-isotype: G1mz; IgG1 mutant-A234, A235; IgG2-isotype: G2m (n-); κ-Km3; And λ.

Fc receptor and C1q studies: mutations in the hinge region (e.g. L234A, L235A) can be used to eliminate inappropriate antibody-dependent cell-mediated cytotoxicity (ADCC) caused by the complexing of antibodies with any over-represented target on the cell membrane And the possibility of complement dependent cytotoxicity (CDC). The presence of these substituted amino acids in the IgG1 hinge region of the mAb is expected to result in reduced binding of the mAb to the human Fc receptor (instead of FcRn) due to the overlap of FcgR binding believed to occur on the IgG1 hinge region Within the site. This feature of mAb can lead to an improved safety profile over antibodies containing wild-type IgG. Binding of mAb to human Fc receptors can be determined by flow cytometry experiments using cell lines (such as THP-1, K562) and engineered CHO cell lines expressing FcgRIIb (or other FcgR). Compared to the IgG1 control monoclonal antibody, mAb displayed reduced binding to FcgRI and FcgRIIa, while binding to FcgRIIb was not affected. C1q triggers a typical complement cascade with subsequent inflammation and / or immunomodulatory responses through the binding and activation of the antigen / IgG immune complex. The C1q binding site on IgG has been mapped to residues in the IgG hinge region. The binding of C1q to increasing concentrations of mAb was assessed by C1q ELISA. As expected when compared to the binding of wild-type control IgG1, the results demonstrated that mAb was unable to bind C1q. In general, mutations in the hinge regions of L234A and L235A eliminated the binding of mAb to FcgRI, FcgRIIa, and C1q, but did not affect the interaction between mAb and FcgRIIb. These data indicate that in vivo, mAbs with mutated Fc normally interact with inhibitory FcgRIIb but may not interact with activated FcgRI and FcgRIIa receptors or C1q.

Human FcRn binding: The neonatal receptor (FcRn) is responsible for transporting IgG across the placenta and controls the catabolic half-life of IgG molecules. It may be necessary to increase the terminal half-life of the antibody to improve efficacy, reduce dosage or frequency of administration, or to improve targeting to a target. Alternatively, it may be advantageous to perform the reverse process, that is, reduce the terminal half-life of the antibody to reduce systemic exposure or improve the target-non-target binding ratio. Adapting the interaction between IgG and its rescue receptor FcRn provides a way to increase or decrease the terminal half-life of IgG. Circulating proteins (including IgG) are dissolved in the fluid phase via microcytotoxicity performed by certain cells, such as those of the vascular endothelium. IgG can bind FcRn in endosomes under slightly acidic conditions (pH 6.0-6.5) and can be recycled to the cell surface where it is released under almost neutral conditions (pH 7.0-7.4). The location of the Fc region binding sites on FcRn80, 16, 17 shows that two histidine residues (His310 and His435) conserved across species determine the pH dependence of this interaction. Using phage presentation technology, mouse Fc region mutations that increase binding to FcRn and extend the half-life of mouse IgG are identified (see Ghetie et al., Nature Biotechnol. , 15 (7): 637-640 (1997)). Mutations in the Fc region that increase the binding affinity of human IgG for FcRn at pH 6.0 instead of pH 7.4 have also been identified (see Dall'Acqua et al. , J. Immunol. , 169 (9): 5171-5180 ( 2002)). In addition, in one case, a similar pH-dependent increase in binding (up to 27-fold) was also observed for rhesus FcRn, and this resulted in a two-fold increase in serum half-life in rhesus monkeys compared to the parental IgG (See Hinton et al., J. Biol. Chem. , 279 (8): 6213-6216 (2004)). The results of these studies indicate that it is feasible to extend the plasma half-life of antibody therapeutics by tailoring the interaction between Fc regions and FcRn. Conversely, mutations in the Fc region that reduce interaction with FcRn can shorten antibody half-life.

B.10. Pharmacokinetics (PK)

To generate a DVD-Ig molecule with a desired pharmacokinetic profile, in one embodiment, a parent mAb with a similar desired pharmacokinetic profile is selected. One consideration is the immunogenic response to individual antibodies (ie, "HAHA", human anti-human antibody response; "HACA", human anti-chimeric antibody response) further complicates the pharmacokinetics of these therapeutic agents . In one embodiment, a monoclonal antibody with minimal or no immunogenicity is used to construct a DVD-Ig molecule so that the resulting DVD-Ig will also have minimal or non-immunogenicity. Some factors that determine the PK of a mAb include, but are not limited to, the inherent properties of the mAb (VH amino acid sequence); immunogenicity; FcRn binding and Fc function.

Because the PK profile in rodents is well correlated with the PK profile of monoclonal antibodies in cynomolgus monkeys and humans (or the PK profile in rodents closely predicts the PK profile of monoclonal antibodies in cynomolgus monkeys and humans), the parent The PK profile of a monoclonal antibody can be easily determined in rodents.

After selecting a parental monoclonal antibody with the desired PK characteristics (and other desired functional properties as discussed herein), DVD-Ig is constructed. Because the DVD-Ig molecule contains two antigen-binding domains from two parental monoclonal antibodies, the PK properties of DVD-Ig were also evaluated. because Therefore, when determining the PK properties of DVD-Ig, a PK assay based on the function of two antigen-binding domains derived from two parental monoclonal antibodies can be used to determine the PK profile. The PK profile of DVD-Ig can be determined. Other factors that can affect the PK profile of DVD-Ig include antigen binding domain (CDR) orientation, linker size, and Fc / FcRn interactions. The PK characteristics of the parental antibodies can be analyzed by assessing the following parameters: absorption, distribution, metabolism, and excretion.

Absorption: To date, the administration of therapeutic monoclonal antibodies has been via parenteral routes (eg intravenous [IV], subcutaneous [SC] or intramuscular [IM]). MAb is absorbed into the systemic circulation from the intercellular space after SC or IM administration, mainly via the lymphatic pathway. Saturable, pre-systemic, proteolytic degradation can lead to variable absolute bioavailability after extravascular administration. In general, since proteolytic capacity is saturated at higher doses, it is observed that absolute bioavailability increases with increasing monoclonal antibody doses. Because lymph fluid is slowly discharged into the vasculature, the absorption process of mAb is usually quite slow, and the duration of absorption can last from several hours to several days. The absolute bioavailability of individual antibodies after SC administration is generally in the range of 50% to 100%. In the case of a transport-mediated structure at the blood-brain barrier (BBB) targeted by the DVD-Ig construct, since the blood-brain barrier (BBB) is in the CNS compartment (where DVD-Ig is released to be able to pass through its The enhanced intercellular transport in the interaction of the two antigen recognition sites) can reduce the circulation time in plasma.

Distribution: After IV administration, monoclonal antibodies usually follow a two-stage serum (or plasma) concentration-time profile, starting with a rapid distribution phase followed by a slow elimination phase. In general, bi-exponential pharmacokinetic models better describe this type of pharmacokinetic profile. The volume of distribution (Vc) of mAb in the central compartment is usually equal to or slightly greater than the volume of plasma (2-3 liters). The different two-stage pattern of serum (plasma) concentration versus time profile may be insignificant for other parenteral routes of administration (such as IM or SC), which is attributed to the distribution phase of the serum (plasma) concentration-time curve by Obscured by longer absorption. Many factors, including physicochemical properties, site-specific and target-targeted receptor-mediated absorption, tissue binding capacity, and mAb dose can affect the biodistribution of mAb. Some of these factors can cause non-linearities in the biodistribution of mAb.

Metabolism and excretion: Due to molecular size, intact monoclonal antibodies are not excreted into the urine through the kidneys. It is primarily inactivated by metabolism (such as catabolism). For IgG-based therapeutic monoclonal antibodies, the half-life is usually in the range of hours or 1-2 days to more than 20 days. The elimination of mAb can be affected by many factors including, but not limited to, affinity for FcRn receptors, immunogenicity of mAb, degree of glycosylation of mAb, sensitivity of mAb to proteolysis, and receptor-mediated Its elimination.

B.11. Tissue cross-reactivity patterns in humans and tox species

The same staining pattern indicates that potential human toxicity can be analyzed in toxicological species. Toxicological species are their animals that study unrelated toxicity.

Select individual antibodies that meet the following two criteria: (1) tissue staining suitable for the known performance of the antibody target; and (2) similar staining patterns from the same organs between human and toxicological species tissues.

Guideline 1: Immune and / or antibody selection usually uses recombinant or synthetic antigens (proteins, carbohydrates or other molecules). Binding to natural counterparts and counterscreens against unrelated antigens are usually part of the screening funnel for therapeutic antibodies. However, screening against multiple antigens is often impractical. Therefore, tissue cross-reactivity studies on human tissues from all major organs are used to exclude improper binding of antibodies to any unrelated antigen.

Guideline 2: Comparative tissue cross-reactivity studies on human and toxicological species tissues (macaques, dogs, possible rodents, and others, such as testing the same 36 or 37 tissues in human studies) can help validate toxicological species Of choice. In a typical tissue cross-reactivity study on frozen tissue sections, therapeutic antibodies can demonstrate predetermined binding to known antigens and / or to a lesser extent based on any low-level interaction (non-specific binding, with similar antigens) Low degree of binding, low degree of interaction based on charge, etc.). In any case, the most relevant toxicological animal species is the one that conforms to the binding of human and animal tissues. The highest degree species.

Tissue cross-reactivity studies follow appropriate regulatory guidelines, including EC CPMP Guideline III / 5271/94 "Production and quality control of mAbs" and 1997 US FDA / CBER "Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use" . Frozen sections (5 μm) of human tissue obtained from autopsies or biopsies were fixed on an objective lens and dried. Peroxidase staining of tissue sections was performed using the avidin-biotin system. FDA's Guidance " Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use ". Related references include Clarke, J. (2004), Boon, L. (2002a), Boon, L. (2002b), Ryan, A. (1999).

Tissue cross-reactivity studies are usually performed in two phases. The first phase includes 32 tissues from a human donor (usually: adrenal, gastrointestinal, prostate, bladder, heart, skeletal muscle, blood cells, kidney, skin, bone marrow, Liver, spinal cord, breast, lung, spleen, cerebellum, lymph nodes, testes, cerebral cortex, ovary, thymus, colon, pancreas, thyroid, endothelium, parathyroid, ureter, eyes, pituitary, uterus, fallopian tube and placenta) . In the second phase, the complete cross-reactivity study used up to 38 tissues (including adrenal glands, blood, blood vessels, bone marrow, cerebellum, brain, cervix, esophagus, eyes, heart, kidneys, Large intestine, liver, lungs, lymph nodes, breast mammary glands, ovaries, fallopian tubes, pancreas, parathyroid, peripheral nerves, pituitary, placenta, prostate, salivary glands, skin, small intestine, spinal cord, spleen, stomach, striated muscle, testes, thymus, thyroid, Tonsils, ureters, bladder and uterus). Studies are usually performed at a minimum of two dose concentrations.

Therapeutic antibodies (i.e., test articles) and isotype control antibodies can be biotinylated for detection of avidin-biotin complex (ABC); other detection methods can include FITC (or other means) labeled Test item tertiary antibody detection, or unlabeled test The test article was pre-complexed with labeled anti-human IgG.

Briefly, frozen sections (about 5 μm) of human tissue obtained from autopsies or biopsies were fixed on an objective lens and dried. Peroxidase staining of tissue sections was performed using the avidin-biotin system. First (in the case of a pre-complex detection system), the test article is incubated with a second biotin-labeled anti-human IgG and forms an immune complex. Immune complexes under test articles at final concentrations of 2 μg / mL and 10 μg / mL were added to the tissue sections on the objective lens and then the tissue sections were reacted with the avidin-biotin-peroxidase kit 30 minute. Subsequently, DAB (3,3'-diaminobenzidine, a substrate for the peroxidase reaction) was applied for 4 minutes for tissue staining. Antigen-agarose beads were used as positive control tissue sections.

Based on the known manifestations of the target antigen in question, any specific staining is judged to be expected (e.g., consistent with antigen performance) or unexpected reactivity. Any stain that is judged to be specific is scored for intensity and frequency. Antigen or serum competition or blocking studies can further assist in determining whether the observed staining is specific or non-specific.

If two selected antibodies are found to meet the selection criteria (appropriate tissue staining, matching staining between human and toxicological animal specific tissues), they can be selected for DVD-Ig generation.

Tissue cross-reactivity studies must be repeated for the final DVD-Ig construct, but although these studies follow the same protocol as outlined herein, since any binding may come from either of the two parent antibodies, and any Illustrated binding needs to be confirmed by complex antigen competition studies, so its evaluation is more complicated.

Obviously, if two parental antibodies are selected for (1) the lack of unexpected tissue cross-reactivity studies and (2) the appropriate similarity of the results of tissue cross-reactivity studies between corresponding human and toxicological animal species tissues, then This greatly simplifies the complex task of performing tissue cross-reactivity studies on multispecific molecules such as DVD-Ig.

B.12. Specificity and selectivity

To generate a DVD-Ig molecule with the required specificity and selectivity, Select a parent mAb with a similar specificity and selectivity profile.

DVD-Ig specific and selective binding studies can be complicated due to four or more binding sites, with two binding sites for each antigen. In short, binding studies on DVD-Ig using ELISA, BIAcore, KinExA, or other interaction studies require monitoring the binding of one, two, or more antigens to the DVD-Ig molecule. Although BIAcore technology can resolve sequential and independent binding of multiple antigens, more traditional methods including ELISA or more modern technologies such as KinExA cannot. Therefore, careful characterization of each parental antibody is critical. After each individual antibody has been specifically characterized, the confirmation of the specific retention of individual binding sites in the DVD-Ig molecule is greatly simplified.

It is obvious that the selection of specificity before combining the two parent antibodies into DVD-Ig greatly simplifies the complicated task of determining the specificity of DVD-Ig.

Antigen-antibody interaction studies can take many forms, including many typical protein-protein interaction studies, including ELISA (enzyme-linked immunosorbent assay), mass spectrometry, chemical cross-linking, SEC with light scattering, equilibrium dialysis, gel permeation , Ultrafiltration, gel chromatography, large area analysis SEC, micropreparative ultracentrifugation (settling balance), spectroscopy, titration microcalorimetry, sedimentation balance (in analytical ultracentrifuge), sedimentation speed (in analytical centrifuge Middle), surface plasmon resonance (including BIAcore). Relevant references include Current Protocols in Protein Science , Volume 3, Chapters 19 and 20 (Coligan et al.) (John Wiley & Sons Inc.) and the references included therein; and Current Protocols in Immunology , ( Coligan et al.) (John Wiley & Sons Inc.) and related references included therein.

Cytokine release in whole blood : The interaction of mAb with human blood cells can be studied by cytokine release assays (Wing et al., Therapeutic Immunol. , 2 (4): 183-190 (1995); Current Protocols in Pharmacology , ( Enna et al. (John Wiley & Sons Inc.); Madhusudan et al., Clin. Cancer Res. , 10 (19): 6528-6534 (2004); Cox et al., Methods , 38 (4): 274-282 (2006); Choi et al., Eur. J. Immunol. , 31 (1): 94-106 (2001)). Briefly, mAbs of various concentrations were incubated with human whole blood for 24 hours. The test concentration should cover a wide range, including the final concentration (including but not limited to 100ng / ml-100μg / ml) that mimics the typical blood content in a patient. After incubation, the presence of IL-1Rα, TNF-α, IL-1b, IL-6 and IL-8 in the supernatant and cell lysates was analyzed. The cytokine concentration profile generated for the mAb was compared to the profile generated by a negative human IgG control and a positive LPS or PHA control. The mAb from both the cell supernatant and the cell lysate showed comparable cytokine profiles to those using control human IgG. In one embodiment, the monoclonal antibody does not interact with human blood cells to spontaneously release inflammatory cytokines.

DVD-Ig's cytokine release study is complicated by four or more binding sites, with two binding sites per antigen. In short, the cytokine release studies described herein measure the effect of complete DVD-Ig molecules on whole blood or other cellular systems, but can analyze which part of the molecule causes cytokine release. Once cytokine release has been detected, the purity of the DVD-Ig preparation must be determined, as some co-purified cellular components can independently lead to cytokine release. If the purity does not cause problems, fragmentation of the DVD-Ig (including (but not limited to) removal of the Fc portion, separation of binding sites, etc.), binding site mutation induction, or other methods may be required to deconvolute any observations product. Obviously, if the choice is made for a lack of cytokine release before the two parent antibodies are combined into a DVD-Ig, this complex task is greatly simplified.

B.13. Cross-reactivity of toxicology studies to other species

In one embodiment, the selected individual antibody has sufficient cross-reactivity with an appropriate toxicological species, such as a cynomolgus monkey. The parent antibody needs to bind to the target of the orthologous species (i.e. cynomolgus monkey) and elicit an appropriate response (regulation, neutralization, activation). In one embodiment, the cross-reactivity (affinity / potency) with orthologous species targets should be within 10 times of the human target. In fact, parental antibodies are evaluated for multiple species (including mice, rats, dogs, monkeys (and other non-human primates)) and disease model species (ie sheep for asthma models). Dear The acceptable cross-reactivity of this monoclonal antibody to toxicological species allows future toxicological studies of DVD-Ig in the same species. For this reason, two parental monoclonal antibodies should have acceptable cross-reactivity to a common toxicological species, thus making it possible to perform DVD-Ig toxicological studies in the same species.

The parental mAb can be selected from a variety of mAbs that are capable of binding specific targets and are well known in the art. These include (but are not limited to) IL-1β, anti-TNF antibodies (US Patent No. 6,258,562), anti-IL-12 and / or anti-IL-12p40 antibodies (US Patent No. 6,914,128), anti-IL-18 antibodies (US Publication No. 2005/0147610 A1), anti-C5, anti-CBL, anti-CD147, anti-gp120, anti-VLA-4, anti-CD11a, anti-CD18, anti-VEGF, anti-CD40L, anti-CD-40 (see, for example, PCT Publication No. WO 2007/124299), anti-Id, anti-ICAM-1, anti-CXCL13, anti-CD2, anti-EGFR, anti-TGF-β2, anti-HGF, anti-cMet, anti-DLL-4, anti-NPR1, anti-PLGF, anti-ErbB3, anti- E-selectin, anti-Fact VII, anti-Her2 / neu, anti-F gp, anti-CD11 / 18, anti-CD14, anti-ICAM-3, anti-RON, anti-CD-19, anti-CD80 (see, for example, PCT Publication No. WO 2003 / 039486), anti-CD4, anti-CD3, anti-CD23, anti-β2-integrin, anti-α4β7, anti-CD52, anti-HLA DR, anti-CD22 (see, e.g., U.S. Patent No. 5,789,554), anti-CD20, anti-MIF, anti-CD64 (FcR), anti-TCRαβ, anti-CD2, anti-Hep B, anti-CA 125, anti-EpCAM, anti-gp120, anti-CMV, anti-gpIIbIIIa, anti-IgE, anti-CD25, anti-CD33, anti-HLA, anti-IGF1,2, anti-IGFR, Anti-VNR integrin, anti-IL-1α, anti-IL-1β, anti- IL-1 receptor, anti-IL-2 receptor, anti-IL-4, anti-IL-4 receptor, anti-IL5, anti-IL-5 receptor, anti-IL-6, anti-IL-6R, RANKL, NGF, DKK , ΑVβ3, anti-IL-8, anti-IL-9, anti-IL-13, anti-IL-13 receptor, and anti-IL-23, IL-23p19 (see Presta, LG, `` Selection, design, and engineering of therapeutic antibodies ", J. Allergy Clin. Immunol. , 116: 731-736 (2005) and the global information website http://www.path.cam.ac.uk/~mrc7/humanisation/antibodies.html ).

The parental mAb can also be selected from a variety of therapeutic antibodies approved for use in clinical trials or for development in clinical use. Such therapeutic antibodies include, but are not limited to, rituximab (Rituxan®, IDEC / Genentech / Roche) (see, e.g., U.S. Patent No. 5,736,137) and are approved for the treatment of non-Hodgkin's lymph Tumor chimeric anti-CD20 antibody; HuMax-CD20, the anti-CD20 currently developed by Genmab, is described in U.S. Patent No. 5,500,362; (Intracel) and PRO70769 (PCT Publication No. WO 2004/056312 (PCT / US2003 / 040426), named "Immunoglobulin Variants and Uses Thereof"); trastuzumab (Herceptin®, Genentech) (see For example, U.S. Patent No. 5,677,171), a humanized anti-Her2 / neu antibody approved for the treatment of breast cancer; Pertuzumab (rhuMab-2C4, Omniarg®), currently developed by Genentech; described in U.S. Patent No. Anti-Her2 antibody in No. 4,753,894; cetuximab (Erbitux®, ImClone) (U.S. Patent No. 4,943,533; PCT Publication No. WO 96/40210), chimeric anti-clinical in clinical trials of various cancers EGFR antibody; ABX-EGF (US special No. 6,235,883), currently developed by Abgenix-Immunex-Amgen; HuMax-EGFr (US No. 10 / 172,317, published as US 2003/0091561, now US Patent No. 7,247,301), currently developed by Genmab; 425, EMD55900, EMD62000 and EMD72000 (Merck KGaA) (U.S. Patent No. 5,558,864; Murthy et al., Arch. Biochem. Biophys. , 252 (2): 773-783 (1991)); ICR62 (Institute of Cancer Research) (PCT Publication No. WO 95/20045; 549-560 (1987); Rodeck et al., J. Cell Biochem., 35 (4): 315-320 (1987); Kettleborough et al., Protein Eng., 4 (7); Modjtahedi et al. Human, J. Cell Biophys. , 22 (1-3): 129-146 (1993); Modjtahedi et al., Br . J. Cancer , 67 (2): 247-253 (1993); Modjtahedi et al., Br. J. Cancer , 73 (2): 228-235 (1996); Modjtahedi et al., Int . J. Cancer , 105 (2): 273-280 (2003)); TheraCIM hR3 (YM Biosciences, Canada, and Centro de Immunologia Molecular, Cuba) (U.S. Patent No. 5,891,996; U.S. Patent No. 6,506,883; Mateo et al., Immunotechnology , 3 (1): 71-81 (1997)); mAb-806 (Ludwig Institue for Cancer Research, Memorial Sloan-K ettering) (Jungbluth et al., Proc. Natl. Acad, Sci. USA., 100 (2): 639-644 (2003)); KSB-102 (KS Biomedix); MR1-1 (IVAX, National Cancer Institute) ( PCT Publication No. WO 01/62931); and SC100 (Scancell) (PCT Publication No. WO 01/88138); alemtuzumab (Campath®, Millenium), currently approved for the treatment of B cells Humanized mAb for chronic lymphocytic leukemia; muromonab-CD3 (Orthoclone OKT3®), an anti-CD3 antibody developed by Ortho Biotech / Johnson &Johnson; ibritumomab tiuxetan (Zevalin®) , Anti-CD20 antibody developed by IDEC / Schering AG; gemtuzumab ozogamicin (Mylotarg®), anti-CD33 (p67 protein) antibody developed by Celltech / Wyeth; afafacept ) (Amevive®), an anti-LFA-3 Fc fusion developed by Biogen; abciximab (ReoPro®), developed by Centocor / Lilly; basiliximab (Simulect®), Developed by Novartis; palivizumab (Synagis®), developed by Medimmune; infliximab (Remicade®), developed by Centocor Anti-TNFα antibody; adalimumab (Humira®), an anti-TNFα antibody developed by Abbott Laboratories; Humicade®, an anti-TNFα antibody developed by Celltech; golimumab (CNTO-148) , A fully human TNF antibody developed by Centocor; etanercept (Enbrel®), a p75 TNF receptor Fc fusion developed by Immunex / Amgen; lenercept, p55TNF previously developed by Roche Receptor Fc fusions; ABX-CBL, an anti-CD147 antibody developed by Abgenix; ABX-IL8, an anti-IL8 antibody developed by Abgenix; ABX-MA1, an anti-MUC18 antibody developed by Abgenix; Pemtumomab (Pemtumomab) ( R1549, 90Y-muHMFG1), anti-MUC1 developed by Antisoma; Therex (R1550), anti-MUC1 antibody developed by Antisoma; AngioMab (AS1405), developed by Antisoma; HuBC-1, developed by Antisoma; Thioplatin (AS1407), Developed by Antisoma; Antegren® (natalizumab), anti-α-4-β-1 (VLA-4) and α-4-β-7 antibodies developed by Biogen; VLA-1 mAb, Anti-VLA-1 integrin antibody developed by Biogen; LTBR mAb, anti-lymphotoxin beta receptor (LTBR) developed by Biogen Antibodies; CAT-152, an anti-TGF-β2 antibody developed by Cambridge Antibody Technology; ABT 874 (J695), an anti-IL-12 p40 antibody developed by Abbott Laboratories; CAT-192, an anti-TGFβ1 developed by Cambridge Antibody Technology and Genzyme Antibodies; CAT-213, an anti-eosinophil chemokine 1 (Eotaxin1) antibody developed by Cambridge Antibody Technology; LymphoStat-B®, an anti-Blys antibody developed by Cambridge Antibody Technology and Human Genome Sciences Inc .; TRAIL-R1mAb , Anti-TRAIL-R1 antibody developed by Cambridge Antibody Technology and Human Genome Sciences, Inc .; Avastin® bevacizumab (rhuMAb-VEGF), anti-VEGF antibody developed by Genentech, and anti-HER antibody developed by Genentech Body family antibodies; anti-tissue factor (ATF), an anti-tissue factor antibody developed by Genentech; Xolair® (Omazumab), an anti-IgE antibody developed by Genentech; Raptiva® (Efalizumab), by Anti-CD11a antibody developed by Genentech and Xoma; MLN-02 antibody (previously LDP-02), developed by Genentech and Millenium Pharmaceuticals; HuMax CD4, developed by Genmab Anti-CD4 antibodies; HuMax-IL15, an anti-IL15 antibody developed by Genmab and Amgen; HuMax-Inflam, developed by Genmab and Medarex; HuMax-Cancer, an anti-heparinase I antibody developed by Genmab and Medarex and Oxford GcoSciences; HuMax- Lymphoma, developed by Genmab and Amgen; HuMax-TAC, developed by Genmab; IDEC-131 and anti-CD40L antibodies developed by IDEC Pharmaceuticals; IDEC-151 (Clenoliximab), anti-CD4 developed by IDEC Pharmaceuticals Antibodies; IDEC-114, an anti-CD80 antibody developed by IDEC Pharmaceuticals; IDEC-152, an anti-CD23 developed by IDEC Pharmaceuticals; anti-macrophage migration factor (MIF) antibody, developed by IDEC Pharmaceuticals; BEC2, an antibody developed by ImClone Individual genotype antibodies; IMC-1C11, an anti-KDR antibody developed by ImClone; DC101, an anti-flk-1 antibody developed by ImClone; an anti-VE cadherin antibody, developed by ImClone; CEA-Cide® (Rabetizumab (labetuzumab)), an anti-carcinoembryonic antigen (CEA) antibody, developed by Immunomedics; LymphoCide® (Epratuzumab), an anti-CD22 antibody developed by Immunomedics; AFP-Cide, by Immunome dics development; MyelomaCide, developed by Immunomedics; LkoCide, developed by Immunomedics; ProstaCide, developed by Immunomedics; MDX-010, anti-CTLA4 antibody developed by Medarex; MDX-060, anti-CD30 antibody developed by Medarex; MDX-070, developed by Developed by Medarex; MDX-018, developed by Medarex; Osidem® (IDM-1) and anti-Her2 antibodies developed by Medarex and Immuno-Designed Molecules; HuMax®-CD4, anti-CD4 antibodies developed by Medarex and Genmab; HuMax-IL15 , Anti-IL15 antibody developed by Medarex and Genmab; CNTO 148, anti-TNFα antibody developed by Medarex and Centocor / Johnson &Johnson; CNTO 1275, anti-cytokine antibody developed by Centocor / Johnson &Johnson; MOR101 and MOR102, by MorphoSys Developed anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibody; MOR201, anti-fibroblast growth factor receptor 3 (FGFR-3) antibody developed by MorphoSys; Nuvion® (vilizumab ( visilizumab)), an anti-CD3 antibody developed by Protein Design Labs; HuZAF®, an anti-gamma interferon antibody developed by Protein Design Labs; anti-α5β1 integrin, developed by Protein Design Labs; anti-IL-12 Developed by Protein Design Labs; ING-1, an anti-Ep-CAM antibody developed by Xoma; Xolair® (Omuzumab), a humanized anti-IgE antibody developed by Genentech and Novartis; and MLN01, an anti-β2 developed by Xoma Integrin antibodies. In another embodiment, the therapeutic agent includes KRN330 (Kirin); huA33 antibody (A33, Ludwig Institute for Cancer Research); CNTO 95 (α V integrin, Centocor); MEDI-522 (α V β3 integrin, Medimmune); Volociximab (α Vβ1 integrin, Biogen / PDL); human mAb 216 (B cell glycosylation epitope, NCI); BiTE MT103 (bispecific CD19 × CD3, Medimmune); 4G7 × H22 (Bispecific B cells × FcγR1, Medarex / Merck KGa); rM28 (bispecific CD28 × MAPG, European Patent No. EP 1 444 268); MDX447 (EMD 82633) (bispecific CD64 × EGFR, Medarex); Catumaxomab (removab) (bispecific EpCAM x anti-CD3, Trion / Fres); Ertumaxomab (bispecific HER2 / CD3, Fresenius Biotech); Austria Orgovomab (OvaRex) (CA-125, ViRexx); Rencarex® (WX G250) (Carbon Anhydrase IX, Wilex); CNTO 888 (CCL2, Centocor); TRC105 (CD105 ( Endothelial factor), Tracon); BMS-663513 (CD137 agonist, Brystol Myers Squibb); MDX-1342 (CD19, Medarex); Siplizumab (MEDI-507) (CD2, M edimmune); Ofatumumab (Humax-CD20) (CD20, Genmab); Rituximab (Rituxan) (CD20, Genentech); veltuzumab (hA20) (CD20, Immunomedics); Epalizumab (CD22, Amgen); Lulimiximab (IDEC 152) (CD23, Biogen); Morozumab-CD3 (CD3, Ortho); HuM291 (CD3 fc Recipient, PDL Biopharma); HeFi-1 (CD30, NCI); MDX-060 (CD30, Medarex); MDX-1401 (CD30, Medarex); SGN-30 (CD30, Seattle Genentics); SGN-33 (Linto Lintuzumab) (CD33, Seattle Genentics); Zanolimumab (HuMax-CD4) (CD4, Genmab); HCD122 (CD40, Novartis); SGN-40 (CD40, Seattle Genentics); Campath1h (Alemtuzumab) (CD52, Genzyme); MDX-1411 (CD70, Medarex); hLL1 (EPB-1) (CD74.38, Immunomedics); Galiximab (IDEC-144) ) (CD80, Biogen); MT293 (TRC093 / D93) (lysed collagen, Tracon); HuLuc63 (CS1, PDL Pharma); ilimimumab (MDX-010) (CTLA4, Brystol Myers Squibb); Tremelimumab (Ticilimuma b), CP-675, 2) (CTLA4, Pfizer); HGS-ETR1 (Mapatumumab) (DR4 TRAIL-R1 agonist, Human Genome Science / Glaxo Smith Kline); AMG-655 ( DR5, Amgen); Apomab (DR5, Genentech); CS-1008 (DR5, Daiichi Sankyo); HGS-ETR2 (lexatumumab) (DR5 TRAIL-R2 agonist, (HGS); Cetuximab (Erbitux) (EGFR, ImClone); IMC-11F8 (EGFR, ImClone); Nitotuzumab (EGFR, YM Bio); Penituzumab Anti (Panitumumab) (Vectabix) (EGFR, Amgen); Zalutumumab (HuMaxEGFr) (EGFR, Genmab); CDX-110 (EGFRvIII, AVANT Immunotherapeutics); Adumab Anti (adecatumumab) (MT201) (Epcam, Merck); edrecolomab (Panorex, 17-1A) (Epcam, Glaxo / Centocor); MORAb-003 (folate receptor a, Morphotech); KW- 2871 (Necrolipid GD3, Kyowa); MORAb-009 (GP-9, Morphotech); CDX-1307 (MDX-1307) (hCGb, Celldex); Trastuzumab (Herceptin) ( HER2, Celldex); Pertuzumab (rhuMAb 2C4) (HER2 (DI), Genentech); Abuzumab (apolizumab) (HLA-DRβ chain, PDL Pharma); AMG-479 (IGF-1R, Amgen); anti-IGF-1R R1507 (IGF1-R, Roche); CP 751871 (IGF1-R, Pfizer); IMC-A12 (IGF1-R, ImClone); BIIB022 (IGF-1R, Biogen); Mik-β-1 (IL-2Rb (CD122), Hoffman LaRoche); CNTO 328 (IL6, Centocor); anti-KIR (1-7F9) ( Killer cell Ig-like receptor (KIR), Novo); Hu3S193 (Lewis (y), Wyeth, Ludwig Institute of Cancer Research); hCBE-11 (LTβR, Biogen); HuHMFG1 (MUC1, Antisoma / NCI); RAV12 (N Linked carbohydrate epitope, Raven); CAL (parathyroid hormone-related protein (PTH-rP), University of California); CT-011 (PD1, CureTech); MDX-1106 (ono-4538) (PD1, Medarex / Ono); MAb CT-011 (PD1, Curetech); IMC-3G3 (PDGFRa, ImClone); bavituximab (phospholipid serine, Peregrine); huJ591 (PSMA, Cornell Research Foundation); muJ591 (PSMA, Cornell Research Foundation); GC1008 (TGFb (pan) inhibitor (IgG4), Genzyme); Infliximab (Remicade) (TNFa, Centocor); A27.15 (transferrin Receptor, Salk Institute, INSERM, PCT company Case No. WO 2005/111082); E2.3 (transferrin receptor, Salk Institute); bevacizumab (Avastin) (VEGF, Genentech); HuMV833 (VEGF, Tsukuba Research Lab, PCT Publication No. WO 2000/034337, University of Texas); IMC-18F1 (VEGFR1, ImClone); IMC-1121 (VEGFR2, ImClone).

C. Build DVD-Ig ^TM Binding protein

Design a multivalent and multispecific dual variable domain immunoglobulin (DVD-Ig ^™ ) binding protein such that two different light chain variable domains (VL) from two different parental monoclonal antibodies are directly Or connected in series via short linkers, followed by the light chain constant domain. Similarly, the heavy chain comprises two different heavy chain variable domains (VHs) connected in series, followed by the constant domains CH1 and the Fc region.

Variable domains can be obtained using recombinant DNA technology from a parental antibody generated by any of the methods described herein. In one embodiment, the variable domain is a murine heavy or light chain variable domain. In another embodiment, the variable domain is a CDR grafted or humanized variable heavy or light chain domain. In one embodiment, the variable domain is a human heavy or light chain variable domain.

In one embodiment, the first and second variable domains are directly linked to each other using recombinant DNA technology. In another embodiment, the variable domains are connected via a linker sequence. In one embodiment, Connect two variable domains. Three or more variable domains can also be linked directly or via a linker sequence. The variable domains may bind the same antigen or may bind different antigens. The DVD-Ig molecule of the present invention may include an immunoglobulin variable domain and a non-immunoglobulin variable domain, such as a ligand binding domain of a receptor and an active domain of an enzyme. DVD-Ig molecules may also contain two or more non-Ig domains.

The linker sequence may be a single amino acid or a linker polypeptide comprising two or more amino acid residues linked by a peptide bond. In one embodiment, the linker sequence is selected from the group consisting of: GGGGSG (SEQ ID NO: 26), GGSGG (SEQ ID NO: 27), GGGGSGGGGS (SEQ ID NO: 28), GGSGGGGSG (SEQ ID NO: 223), GGSGGGGSGS (SEQ ID NO: 29), GGSGGGGSGGGGS (SEQ ID NO: 30), GGGGSGGGGSGGGG (SEQ ID NO: 31), GGGGSGGGGSGGGGS (SEQ ID NO: 32), ASTKGP (SEQ ID NO: 33), ASTKGPSVFPLAP ( (SEQ ID NO: 34), TVAAP (SEQ ID NO: 35), RTVAAP (SEQ ID NO: 224), TVAAPSVFIFPP (SEQ ID NO: 36), RTVAAPSVFIFPP (SEQ ID NO: 225), AKTTPKLEEGEFSEAR (SEQ ID NO: 37) ), AKTTPKLEEGEFSEARV (SEQ ID NO: 38), AKTTPKLGG (SEQ ID NO: 39), SAKTTPKLGG (SEQ ID NO: 40), SAKTTP (SEQ ID NO: 41), RADAAP (SEQ ID NO: 42), RADAAPTVS (SEQ ID NO: 43), RADAAAAGGPGS (SEQ ID NO: 44), RADAAAAGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 45), SAKTTPKLEEGEFSEARV (SEQ ID NO: 46), ADAAP (SEQ ID NO: 47), ADAAPTVSIFPP (SEQ ID NO: 48) , QPKAAP (SEQ ID NO: 49), QPKAAPSVTLFPP (SEQ ID NO: 50), AKTTPP (SEQ ID NO: 51), AKTTPPSVTPLAP (SEQ ID NO: 52), AKTTAP (SEQ ID NO: 53), AKTTAPSVYPL AP (SEQ ID NO: 54), GENKVEYAPALMALS (SEQ ID NO: 55), GPAKELT PLKEAKVS (SEQ ID NO: 56), and GHEAAAVMQVQYPAS (SEQ ID NO: 57). The selection of the linker sequence is based on the crystal structure analysis of several Fab molecules. There is a natural flexible linkage between the variable domain in the Fab or antibody molecular structure and the CH1 / CL constant domain. This natural linkage contains about 10-12 amino acid residues, which are provided by 4-6 residues from the C-terminus of the V domain and 4-6 residues from the N-terminus of the CL / CH1 domain. The DVD-Ig described herein can use 5-6 amino acid residues or 11-12 amino acid residues at the N-terminus of CL or CH1 as the linkers in the light and heavy chains of DVD-Ig, respectively. generate. The N-terminal residues of the CL or CH1 domains, especially the first 5-6 amino acid residues, adopt a ring configuration that does not have a strong secondary structure, and therefore can serve as a flexible linker between two variable domains. Because the N-terminal residues of the CL or CH1 domain are part of the Ig sequence, the N-terminal residues of the CL or CH1 domain are a natural extension of the variable domain, and thus to a large extent make potential self-linkers and junctions appear. Minimize any immunogenicity.

Other linker sequences may include any sequence of any length of the CL / CH1 domain, but not all residues of the CL / CH1 domain; for example, 5-12 amino acid residues before the CL / CH1 domain; light chain linkers may From Cκ or Cλ; and the heavy chain linker can be derived from CH1 of any isotype, including Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ. Linker sequences can also be derived from other proteins, such as Ig-like proteins (eg, TCR, FcR, KIR); G / S-based sequences; hinge domain-derived sequences; and other natural sequences from other proteins.

In one embodiment, the constant domain is linked to two linked variable domains using recombinant DNA technology. In one embodiment, a sequence comprising a tandemly linked heavy chain variable domain is connected to a heavy chain constant domain and a sequence comprising a tandemly linked light chain variable domain is connected to a light chain constant domain. In one embodiment, the constant domains are a human heavy chain constant domain and a human light chain constant domain, respectively. In one embodiment, the DVD heavy chain is further linked to the Fc region. The Fc region may be a native sequence Fc region or a variant Fc region. In another embodiment, the Fc region is a human Fc region. In another embodiment, the Fc region comprises an Fc region from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

In a preferred embodiment, two heavy chain DVD polypeptides and two light chain DVD polypeptides are combined to form a DVD-Ig molecule. A detailed description of specific DVD-Ig molecules capable of binding to a specific target antigen, such as IL-1β, and methods of making the same are provided in the Examples section below.

D. Production of DVD-Ig binding protein

The DVD-Ig binding protein of the present invention can be produced by any of a number of techniques known in the art, such as expression from a host cell, wherein the DVD-Ig heavy chain and DVD-Ig light are encoded by standard techniques. The strand expression vector is transfected into a host cell. The various forms of the term "transfection" are intended to encompass a variety of techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-polyglucose transfection, and the like. Although the DVD-Ig protein of the present invention can be expressed in prokaryotic or eukaryotic host cells, since eukaryotic cells (and especially mammalian cells) are more likely to assemble and secrete DVDs that are properly folded and immunocompetent than prokaryotic cells -Ig protein, and thus DVD-Ig protein is expressed in such eukaryotic cells (eg, mammalian host cells).

Exemplary mammalian host cells expressing the recombinant antibodies of the present invention include Chinese hamster ovary (CHO cells) (including dhfr-CHO cells in Urlaub and Chain, Proc. Natl. Acad. Sci. USA , 77: 4216-4220 (1980 ), Which is used with DHFR selectable markers, for example, as described in Kaufman and Sharp, J. Mol. Biol. , 159: 601-621 (1982)), NSO myeloma cells, COS cells, SP2 and PER. C6 cells. When a recombinant expression vector encoding a DVD-Ig protein is introduced into a mammalian host cell, the DVD-Ig protein is sufficient to allow the expression of the DVD-Ig protein in the host cell or the secretion of the DVD-Ig protein to the host by culturing the host cell. Cells are produced over time in the medium in which they are grown. DVD-Ig protein can be recovered from the culture medium using standard protein purification methods.

In an exemplary system that recombinantly expresses the DVD-Ig protein of the present invention, a recombinant expression vector encoding both the DVD-Ig heavy chain and the DVD-Ig light chain is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. . Within the recombinant expression vector, the DVD-Ig heavy chain and light chain genes are each operably linked to the CMV enhancer / AdMLP promoter regulatory module to drive the genes High degree of transcription. The recombinant expression vector also carries the DHFR gene, which allows the use of methotrexate selection / amplification to select CHO cells that have been transfected with the vector. The selected transformant host cells were cultured to allow expression of the DVD-Ig heavy and light chains and recovery of the complete DVD-Ig protein from the culture medium. Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select transformants, cultivate host cells, and recover DVD-Ig protein from the culture medium. In addition, the present invention provides a method for synthesizing the DVD-Ig protein of the present invention by culturing the host cell of the present invention in a suitable medium until the DVD-Ig protein of the present invention is synthesized. The method may further include isolating the DVD-Ig protein from the culture medium.

An important feature of DVD-Ig is that it can be produced and purified in a manner similar to conventional antibodies. The production of DVD-Ig results in a homogeneous, single main product with the required dual specific activity without any sequence modification or any kind of chemical modification of the constant region. The other methods described previously to generate "bispecific", "multispecific" and "multispecific multivalent" full-length binding proteins do not produce a single primary product, but actually result in assembled inactive, monospecific, multispecific A mixture of specific, multivalent, full-length binding proteins and multivalent full-length binding proteins with combinations of different binding sites is produced intracellularly or secreted. As an example, based on the design described by Miller and Presta (PCT Publication No. WO 2001/077342), there are 16 possible combinations of heavy and light chains. Therefore, only 6.25% of the protein may be in the desired active form, rather than in the form of a single main product or a single primary product compared to the other 15 possible combinations. The separation of desired, fully active forms of proteins from inactive and partially active forms of proteins using standard chromatography techniques commonly used in large-scale manufacturing remains to be proven.

Surprisingly, the design of the "dual-specific multivalent full-length binding protein" of the present invention results in the assembly of the dual variable domain light chain and the dual variable domain heavy chain mainly into the desired "dual-specific multivalent full-length binding protein".

At least 50%, at least 75%, and at least 90% of the assembled and expressed DVD-Ig molecules are the desired dual-specific tetravalent protein. This aspect of the invention particularly enhances the commercial effectiveness of the invention use. Therefore, the present invention includes a method of expressing a dual variable domain light chain and a dual variable domain heavy chain in a single cell, thereby generating a single primary product of a "dual-specific tetravalent full-length binding protein".

The invention provides a method for expressing a dual variable domain light chain and a dual variable domain heavy chain in a single cell, thereby generating a "primary product" of a "dual-specific tetravalent full-length binding protein", wherein the "primary product" comprises More than 50% of all assembled proteins of dual variable domain light chain and dual variable domain heavy chain.

The present invention provides a method for expressing a dual variable domain light chain and a dual variable domain heavy chain in a single cell, thereby generating a single "primary product" of a "dual-specific tetravalent full-length binding protein", wherein the "primary product" is More than 75% of all assembled proteins including dual variable domain light chains and dual variable domain heavy chains.

The present invention provides a method for expressing a dual variable domain light chain and a dual variable domain heavy chain in a single cell, thereby generating a single "primary product" of a "dual-specific tetravalent full-length binding protein", wherein the "primary product" is More than 90% of all assembled proteins including dual variable domain light chains and dual variable domain heavy chains.

6. Production of IL-1β binding protein and cell lines producing binding protein

The IL-1β binding proteins (including anti-IL-1β antibodies) of the present invention preferably exhibit a higher ability to reduce or neutralize IL-1β activity, for example, as in several in vitro and in vivo assays known in the art Evaluated by either. The IL-1β binding protein of the present invention preferably also exhibits a higher ability to reduce or neutralize IL-1β activity.

In a preferred embodiment, the binding protein or antigen-binding portion thereof binds human IL-1β, wherein the binding protein or antigen-binding portion thereof has a k _off rate constant (such as a surface plasma) of about 0.1s ^-1 or less than 0.1s ^-1 . the measured resonance) from human IL-1β dissociated, or about 1 × 10 ^-6 M or 1 × 10 ^-6 M or less of IC ₅₀ inhibition of human IL-1β activity. Alternatively, the binding protein or its antigen-binding portion can be dissociated from human IL-1β at a k _off rate constant (as measured by surface plasmon resonance) below about 1 × 10 ^-2 s ^-1 or 1 × 10 ^-2 s ^-1 . or may be about 1 × 10 ^-7 M or 1 × 10 ^-7 M IC ₅₀ of the inhibition of human IL-1β activity. Alternatively, the binding protein or its antigen-binding portion can be dissociated from human IL-1β at a k _off rate constant (as measured by surface plasmon resonance) below about 1 × 10 ^-3 s ^-1 or 1 × 10 ^-3 s ^-1 . or may be about 1 × 10 ^-8 M or 1 × 10 ^-8 M IC ₅₀ of the inhibition of human IL-1β. Alternatively, the binding protein or its antigen-binding portion can be dissociated from human IL-1β at a k _off rate constant (as measured by surface plasmon resonance) below about 1 × 10 ^-4 s ^-1 or 1 × 10 ^-4 s ^-1 . or may be about 1 × 10 ^-9 M or 1 × 10 ^-9 M IC ₅₀ of the inhibition of human IL-1β activity. Alternatively, the binding protein or its antigen-binding portion can be dissociated from human IL-1β at a k _off rate constant (as measured by surface plasmon resonance) below about 1 × 10 ^-5 s ^-1 or 1 × 10 ^-5 s ^-1 . or may be about 1 × 10 ^-10 M or less 1 × 10 ^-10 M IC ₅₀ of inhibition of the activity of human IL-1β. Alternatively, the binding protein or its antigen-binding portion can be dissociated from human IL-1β at a k _off rate constant (as measured by surface plasmon resonance) below about 1 × 10 ^-5 s ^-1 or 1 × 10 ^-5 s ^-1 . or may be about 1 × 10 ^-11 M or less 1 × 10 ^-11 M IC ₅₀ of inhibition of the activity of human IL-1β.

In certain embodiments, the binding protein comprises a heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region. The heavy chain constant region is preferably an IgG1 heavy chain constant region or an IgG4 heavy chain constant region. In addition, the antibody may comprise a light chain constant region (a kappa light chain constant region or a lambda light chain constant region). The antibody preferably comprises a kappa light chain constant region. Alternatively, the antibody portion may be, for example, a Fab fragment or a single-chain Fv fragment.

Substitution of amino acid residues in the Fc portion to alter antibody effector functions is known in the art (Winter et al., U.S. Pat. Nos. 5,648,260 and 5,624,821). The Fc portion of an antibody mediates several important effector functions, such as cytokine induction, ADCC, phagocytosis, complement-dependent cytotoxicity (CDC), and half-life / clearance of antibodies and antigen-antibody complexes. Depending on the goal of treatment, these effector functions are required for therapeutic antibodies in some cases, but may be unnecessary or even harmful in other cases. Certain human IgG isotypes, especially IgG1 and IgG3, mediate ADCC and CDC via binding to FcγR and complement C1q, respectively. Neonatal Fc receptor (FcRn) is a key component that determines the circulating half-life of antibodies. In another embodiment, at least one of the constant regions of an antibody (e.g., the Fc region of an antibody) is replaced. Amino acid residues to alter the effector function of the antibody.

An embodiment provides a labeled binding protein in which an antibody or antibody portion of the invention is derivatized or linked to another functional molecule, such as another peptide or protein. For example, a labeled binding protein of the present invention can function with one or more other molecular entities by allowing an antibody or antibody portion of the present invention (by chemical coupling, genetic fusion, non-covalent association, or other means) Sexual connection for derivatization, the molecular entity or entities are, for example, another antibody (eg, a bispecific antibody or a bifunctional antibody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and / or an antibody or antibody A protein or peptide partially associated with another molecule, such as a streptavidin core region or a polyhistidine tag.

Detectable agents suitable for derivatizing a binding protein (such as an antibody or antibody portion) of the invention include fluorescent compounds. Exemplary fluorescently detectable agents include luciferin, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthosulfonyl chloride, phycoerythrin, and the like. Antibodies can also be derivatized with detectable enzymes such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like. When the antibody is derivatized with a detectable enzyme, it is detected by adding other reagents that the enzyme uses to generate a detectable reaction product. For example, when a detectable agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine produces a colored reaction product that is detectable. The antibody can also be derivatized with biotin and detected by indirect measurement of the binding of avidin or streptavidin.

Another embodiment of the present invention provides a crystallized binding protein. The invention preferably relates to crystals of intact anti-IL-1β antibodies and fragments thereof as disclosed herein, as well as formulations and compositions comprising such crystals. In one embodiment, the crystallized binding protein has a longer in vivo half-life compared to the soluble counterpart of the binding protein. In another embodiment, the binding protein retains biological activity after crystallization.

The crystalline binding protein of the present invention can be prepared according to methods known in the art and as disclosed in PCT Publication No. WO 02/072636, which is incorporated by reference. In this article.

Another embodiment of the present invention provides a glycosylation binding protein, wherein the antibody or antigen-binding portion thereof comprises one or more carbohydrate residues. Early in vivo protein production can undergo further processing called post-translational modification. In particular, sugar (glycosyl) residues can be added enzymatically, a method called glycosylation. The resulting protein with a covalently linked oligosaccharide side chain is called a glycosylated protein or glycoprotein.

Naturally occurring antibodies are glycoproteins that have one or more carbohydrate residues in the Fc domain and the variable domain. Carbohydrate residues in the Fc domain have an important effect on the effector function of the Fc domain and have the least effect on the antigen binding or half-life of the antibody (Jefferis, R., Biotechnol. Prog. , 21: 11-16 (2005)). In contrast, glycosylation of variable domains can have an effect on the antigen-binding activity of antibodies. Glycosylation in the variable domain may be attributed to steric hindrance and may have a negative effect on antibody binding affinity (Co et al., Mol. Immunol. , 30: 1361-1367 (1993)), or result in increased affinity for the antigen (Wallick et al. , J. Exp. Med. , 168: 1099-1109 (1988); Wright et al., EMBO J. , 10: 2717-2723 (1991)).

One aspect of the present invention relates to the generation of a glycosylation site mutant in which the O or N-linked glycosylation site of the binding protein has been mutated. Those skilled in the art can generate such mutants using standard well-known techniques. Glycation site mutants that retain biological activity but have increased or decreased binding activity are another object of the present invention.

In another embodiment, the glycosylation of an antibody or antigen-binding portion of the invention is modified. For example, non-glycosylated antibodies can be made (ie, the antibodies lack glycosylation). Glycosylation can be altered to, for example, increase the affinity of an antibody for an antigen. Such carbohydrate modifications can be achieved, for example, by changing one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made, resulting in elimination of one or more variable region glycosylation sites to thereby eliminate glycosylation at that site. This non-glycosylation can increase the affinity of the antibody for the antigen. This method is described in further detail in PCT Publication No. WO 2003/016466 and the United States National Patent Nos. 5,714,350 and 6,350,861.

Alternatively or in addition, modified binding proteins of the invention can be made that have altered types of glycosylation, such as low fucosylated antibodies with reduced amounts of fucosyl residues (see Kanda et al., J. Biotechnol. , 130 (3): 300-310 (2007)) or antibodies with an increased bisected GlcNAc structure. These altered glycosylation patterns have been shown to increase the ADCC capabilities of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation mechanism. Cells with altered glycosylation mechanisms have been described in the art and can be used as host cells by which to express the recombinant antibodies of the invention to thereby produce antibodies with altered glycosylation. See, for example, Shields et al., J. Biol. Chem. , 277: 26733-26740 (2002); Umana et al., "Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity", Nat. Biotechnol. , 17: 176-180 (1999), and European Publications EP 1 176 195; PCT Publications WO 03/035835 and WO 99/54342.

Protein glycosylation depends on the amino acid sequence of the relevant protein and the host cell that expresses the protein. Different organisms can produce different glycosylases (such as glycosyltransferases and glycosidases) and have different available substrates (nucleotide sugars). Due to these factors, protein glycosylation patterns and the composition of glycosyl residues may vary depending on the host system that represents a particular protein. Glycosyl residues suitable for use in the present invention may include, but are not limited to, glucose, galactose, mannose, fucose, N-acetylglucosamine and sialic acid. The glycosylation-binding protein preferably comprises a glycosyl residue that renders the glycosylation mode to a human mode.

Those skilled in the art know that different protein glycosylation can produce different protein characteristics. For example, the efficacy of a therapeutic protein produced in a microbial host (such as yeast) and glycosylated using a yeast endogenous pathway can be reduced compared to the same protein expressed in mammalian cells (such as a CHO cell line) . These glycoproteins can also be immunogenic in humans and exhibit reduced half-lives in vivo after administration. Characteristics of humans and other animals Heterosexual receptors recognize specific glycosyl residues and promote rapid clearance of proteins from the bloodstream. Other adverse effects may include changes in protein folding, solubility, sensitivity to proteases, transport, transport, compartmentalization, secretion, recognition by other proteins or factors, antigenicity or allergenicity. Therefore, practitioners may prefer therapeutic proteins with specific glycosylation compositions and patterns, such as those that are the same or at least similar to those produced in human cells or in species-specific cells of predetermined individual animals .

A glycosylated protein that behaves differently from the glycosylated protein of the host cell can be achieved by genetically modifying the host cell to express a heterologous glycosylase. Using techniques known in the art, practitioners can produce antibodies or antigen-binding portions thereof that exhibit glycosylation of human proteins. For example, yeast strains have been genetically modified to express non-naturally occurring glycosylation enzymes such that the glycosylated proteins (glycoproteins) produced in these yeast strains exhibit the same proteins as animal cells, especially human cells Glycosylation (US Publication Nos. 2004/0018590 and 2002/0137134).

In addition to binding proteins, the invention also relates to anti-idiotypic (anti-Id) antibodies specific for the binding proteins of the invention. An anti-Id antibody is an antibody that recognizes a unique determinant that is usually associated with the antigen-binding region of another antibody. Anti-Id can be prepared by immunizing an animal with a binding protein or its CDR-containing region. The immunized animal recognizes and responds to the genotype determinant of the immune antibody and produces an anti-Id antibody. Obviously, it is easier to generate anti-idiotypic antibodies that incorporate two or more parental antibodies into a DVD-Ig molecule; and binding is confirmed by methods recognized in the art (e.g., BIAcore, ELISA) Studies have been performed to examine that anti-idiotypic antibodies specific to the genotype of each parental antibody also recognize the genotype (eg, antigen-binding site) of the genotype in the case of DVD-Ig. Anti-idiotypic antibodies specific to each of two or more antigen-binding sites of DVD-Ig provide an ideal reagent for measuring the DVD-Ig concentration of human DVD-Ig in the serum of patients. For example, a DVD-Ig concentration assay can be established using a "sandwich assay ELISA format" in which antibodies to the first antigen-binding region are coated on a solid phase (Such as BIAcore wafers, ELISA disks, etc.), rinse with washing buffer, incubate with the serum sample, another rinse step and finally incubate with another anti-idiotypic antibody of another antigen binding site, itself by enzyme Label to quantify the binding reaction. In one embodiment, for a DVD-Ig with two or more different binding sites, the anti-idiotypic antibodies of the two outermost binding sites (farthest and closest to the constant region) will not only assist in the determination of The DVD-Ig concentration also proves the integrity of the molecule in vivo. Each anti-Id antibody can also be used as an "immunogen" to induce an immune response in another animal, producing so-called anti-anti-Id antibodies.

In addition, those skilled in the art should understand that a host cell library that has been genetically engineered to express various glycosylation enzymes can be used to express related proteins, so that member host cells of the library can produce related proteins with variant glycosylation patterns. . Practitioners can then select and isolate related proteins with specific novel glycosylation patterns. Proteins with a novel selected glycosylation pattern preferably exhibit improved or altered biological properties.

7. Uses of IL-1β binding protein

Assuming that the IL-1β binding protein or antigen-binding portion thereof of the present invention has the ability to bind human IL-1β, it can be used to use conventional immunoassays such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or Tissue immunohistochemistry) to detect IL-1β (for example in biological samples such as serum or plasma). The present invention provides a method for detecting IL-1β in a biological sample, which comprises contacting a biological sample with a binding protein or antigen-binding portion of the present invention and detecting the binding protein (or antigen-binding portion) or non-binding protein that binds to IL-1β. The bound binding protein (or binding moiety) is used to detect IL-1β in the biological sample. The binding protein is directly or indirectly labeled with a detectable substance to help detect bound or unbound antibodies. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; examples of suitable prosthetic complexes include streptavidin / biotin and anti- Biotin protein / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, sulfanil Chlorine or phycoerythrin; examples of luminescent materials include luminol; and examples of suitable radioactive materials include ³ H, ¹⁴ C, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I, ¹⁷⁷ Lu, ¹⁶⁶ Ho or ¹⁵³ Sm.

Instead of labeled binding proteins, human IL-1β in biological fluids can be assayed by competitive immunoassays using rh IL-1β standards labeled with detectable substances and unlabeled human IL-1β binding proteins. In this assay, a biological sample, a labeled rh IL-1β standard, and a human IL-1β binding protein are combined and the amount of labeled rh IL-1β standard bound to an unlabeled antibody is determined. The amount of human IL-1β in a biological sample is inversely proportional to the amount of labeled rh IL-1β standard bound to the IL-1β binding protein. Similarly, human IL-1β in biological fluids can also be assayed by competitive immunoassays using rh IL-1β standards labeled with detectable substances and unlabeled human IL-1β binding proteins.

The binding protein and IL-1β binding portion of the present invention are preferably capable of neutralizing human IL-1β activity in vitro and in vivo. Thus, the binding proteins of the present invention and their IL-1β binding portions can be used, for example, in cell cultures containing human IL-1β, in human individuals, or in other mammals with IL-1β cross-reacting with the antibodies of the present invention Inhibition of human IL-1β activity in animal individuals. In one embodiment, the present invention provides a method for inhibiting human IL-1β activity, which comprises contacting human IL-1β with the IL-1β binding protein or a binding portion thereof of the present invention to inhibit human IL-1β activity. For example, in a cell culture containing or suspected of containing human IL-1β, the IL-1β binding protein or a binding portion thereof of the present invention can be added to a culture medium to inhibit human IL-1β activity in the culture.

In another embodiment, the present invention provides a method for reducing human IL-1β activity in an individual, preferably from an individual suffering from a disease or condition in which IL-1β activity is harmful. The present invention provides a method for reducing IL-1β activity in an individual suffering from the disease or condition, which method comprises administering to the individual an antibody or antibody portion of the present invention such that the IL-1β activity in the individual is reduced. IL-1β is preferably human IL-1β and the individual is a human individual. Alternatively, the individual may Shows that mammals can bind IL-1β to antibodies. In addition, the individual may be a mammal to which IL-1β has been introduced (eg, by administration of IL-1β or by expression of an IL-1β transgenic gene). The IL-1β binding protein of the present invention can be administered to a human individual for therapeutic purposes. In addition, the binding protein of the present invention can be administered to a non-human mammal expressing IL-1β capable of binding to the antibody for veterinary purposes or as an animal model of human disease. With regard to the latter, these animal models can be used to assess the therapeutic efficacy of the antibodies of the invention (e.g., testing the dosage and time course of administration).

The term "IL-1β activity is a deleterious condition" as used herein is intended to include diseases and other conditions in which the presence of IL-1β in an individual suffering from the condition has been demonstrated or is suspected to cause pathophysiological abnormalities of the condition or to contribute to the deterioration of the condition . Thus, a condition in which IL-1β activity is deleterious is a condition in which a decrease in IL-1β activity is expected to reduce the symptoms and / or progression of the condition. Such conditions can be confirmed, for example, by an increase in the concentration of IL-1β in the biological fluid of the individual suffering from the condition (e.g., an increase in the concentration of IL-1β in the serum, plasma, synovial fluid, etc. of the individual), which can be, for example, using Anti-IL-1β antibody to detect. Non-limiting examples of conditions that can be treated with the antibodies of the invention include those conditions discussed below in the section on pharmaceutical compositions of the antibodies of the invention.

The DVD-Ig of the present invention can bind only IL-1β or multiple antigens (eg, human IL-1β and another non-IL-1β antigen). Therefore, DVD-Ig can block or reduce the activity of hu IL-1β and the activity of another target antigen. Such other target antigens may include soluble targets (such as IL-la) and cell surface receptor targets (such as VEGFR, EGFR).

These other antigens include, but are not limited to, the targets listed in the following public databases, which include databases available through the World Wide Web and incorporated herein by reference. These target databases include: therapeutic targets (http://xin.cz3.nus.edu.sg/group/cjttd/ttd.asp); cytokines and cytokine receptors (http: // www. cytokinewebfacts.com/, http://www.copewithcytokines.de/cope.cgi, and http://cmbi.bjmu.edu.cn/cmbidata/cgf/CGF_Database/ cytokine.medic.kumamoto-u.ac.jp/ CFC / indexR.html); chemokines (http://cytokine.medic.kumamoto-u.ac.jp/CFC/CK/Chemokine.html); chemokine receptors and GPCRs (http: // csp. medic.kumamoto-u.ac.jp/CSP/Receptor.html, http://www.gpcr.org/7tm/); olfactory receptors (http://senselab.med.yale.edu/senselab/ORDB/ default.asp); receptor (http://www.iuphar-db.org/iuphar-rd/list/index.htm); cancer target (http://cged.hgc.jp/cgi-bin/input .cgi); secreted proteins as potential antibody targets (http://spd.cbi.pku.edu.cn/); protein kinases (http://spd.cbi.pku.edu.cn/), and Human CD markers (http://content.labvelocity.com/tools/6/1226/CD_table_final_locked.pdf) and (Zola et al., "CD molecules 2005: human cell differentiation molecules", Bl ood, 106: 3123-3126 (2005) ).

DVD-Ig is suitable as a therapeutic agent to block two or more different targets simultaneously (i.e. human IL-1β and one or more other non-IL-1β target antigens) to enhance efficacy / safety and / or increase Patient coverage. Such targets may include soluble targets (TNF) and cell surface receptor targets (VEGFR and EGFR).

In addition, the DVD-Ig of the present invention can be used for tissue-specific delivery (targeting tissue markers and disease mediators to enhance local PK, thus achieving higher efficacy and / or lower toxicity), including intracellular delivery (targeting internal Receptors and intracellular molecules) to the brain (targeting transferrin receptors and CNS disease mediators to cross the blood-brain barrier). DVD-Ig can also serve as a carrier protein that delivers an antigen to a specific location and increases the half-life of the antigen via a non-neutralizing epitope that binds that antigen. In addition, DVD-Ig can be designed to physically connect to or target medical devices implanted in patients (see Burke et al., "Zotarolimus eluting stents", Adv. Drug Deliv. Rev., 58 (3) : 437-446 (2006); Hildebrand et al., "Surface coatings for biological activation and functionalization of medical devices", Surface and Coatings Technology , 200 (22-23): 6318-6324 (2006); Wu et al., "Drug / device combinations for local drug therapies and infection prophylaxis ", Biomaterials , 27: 2450-2467 (2006); Marques et al.," Mediation of the Cytokine Network in the Implantation of Orthopedic Devices ", Chapter 21, Biodegradable Systems in Tissue Engineering and Regenerative Medicine , (edited by Reis et al.) (CRC Press LLC, Boca Raton, 2005) pp. 377-397). In short, directing an appropriate type of cell to the site of a medical implant can promote healing and recovery of normal tissue function. Alternatively, mediators (including, but not limited to, cytokines) that inhibit the release of a device after device implantation by DVD-Ig coupled to the device or targeted to the device are also provided. For example, vascular stents have been used in interventional cardiology for many years to clear blocked arteries and improve blood flow to the heart muscle. However, it is known that conventional bare metal vascular stents cause restenosis (re-narrowing of arteries in treated areas) and can produce blood clots in some patients. Recently, anti-CD34 antibody-coated vascular stents that reduce restenosis and prevent the occurrence of blood clots by capturing endothelial progenitor cells (EPC) circulating throughout the blood have been described. Endothelial cells are cells lined with blood vessels that allow blood to flow smoothly. EPC adheres to the hard surface of the vascular stent, forming a smooth layer that not only promotes healing but also prevents restenosis and blood clots (complications previously associated with the use of vascular stents) (Aoki et al., J. Am. Coll. Cardiol. , 45 ( 10): 1574-1579 (2005)). In addition to improving outcomes for patients requiring vascular stents, it also has implications for patients requiring cardiovascular bypass surgery. For example, an anti-EPC antibody-coated repair blood vessel (artificial artery) eliminates the need to use arteries from a patient's leg or arm for bypass grafting. This will reduce surgery and anesthesia time, which in turn will reduce coronary surgery deaths. DVD-Ig in a manner that allows it to bind to cell surface markers (such as CD34) and proteins (or any kind of epitope, including, but not limited to, proteins, lipids, and polysaccharides) that have been coated on implanted devices Designed to facilitate cell recruitment. These methods are also generally applicable to other medical implants. Alternatively, the DVD-Ig can be coated on a medical device and after all DVDs are implanted and released from the device (or any other need that may require other fresh DVD-Ig, including aging and denaturation of the loaded DVD-Ig) The device can be reloaded by systemically administering fresh DVD-Ig to a patient, where DVD-Ig is designed to bind to related targets (cytokines, cell surface markers (such as CD34) through a set of binding sites). Etc.) and bound to the coated target (including any kind of protein, epitope, including (but not limited to) lipids, polysaccharides and polymers) by another set of binding sites. This technique has the advantage of expanding the applicability of coated implants.

A. Use of DVD-Ig in various diseases

The DVD-Ig molecules of the present invention are also suitable as therapeutic molecules for treating various diseases. The DVD molecules can bind one or more targets related to a particular disease. Examples of such targets in various diseases are described below.

Human autoimmune and inflammatory response

In one aspect, the DVD-Ig binding protein of the present invention is capable of binding human IL-1β and one or more antigens involved in general autoimmune and inflammatory responses, including C5, CCL1 (I-309), CCL11 (eosinophilic) Granulocyte chemokine), CCL13 (mcp-4), CCL15 (MIP-1d), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19, CCL2 (mcp-1), CCL20 (MIP -3a), CCL21 (MIP-2), CCL23 (MPIF-1), CCL24 (MPIF-2 / eosinophil chemokine-2), CCL25 (TECK), CCL26, CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (mcp-3), CCL8 (mcp-2), CXCL1, CXCL10 (IP-10), CXCL11 (I-TAC / IP-9), CXCL12 (SDF1), CXCL13, CXCL14, CXCL2, CXCL3, CXCL5 (ENA-78 / LIX), CXCL6 (GCP-2), CXCL9, IL13, IL8, CCL13 (mcp-4), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7 , CCR8, CCR9, CX3CR1, IL8RA, XCR1 (CCXCR1), IFNA2, IL10, IL13, IL17C, IL1A, IL1B, IL1F10, IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, IL22, IL5, IL8, IL9, LTA, LTB, MIF, SCYE1 (endothelial monocyte-activated cytokine), SPP1, TNF, TNFSF5, IFNA2, IL10RA, IL10RB, IL13, IL13RA1, IL5RA, IL9, IL9R, ABCF1, BCL6, C3, C4A, CEBPB, CRP, ICEBERG, IL1R1, IL1RN, IL8RB, LTB4R, TOLLIP, FADD, IRAK1, IRAK2, MYD88, NCK2, TNFAIP3, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, CD28, CD3E, CD3G, CD3Z, CD69, CD80, CD86, CNR1, CTLA4, CYSLTR1, FCER1A, FCER2, FCGR3A, GPR44, HAVCR2, OPRD1, P2RX7, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, BLR1, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CL1, CX3CR1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL10, CXCL12, CXCL11 CXCR4, GPR2, SCYE1, SDF2, XCL1, XCL 2, XCR1, AMH, AMHR2, BMPR1A, BMPR1B, BMPR2, C19orf10 (IL27w), CER1, CSF1, CSF2, CSF3, DKFZp451J0118, FGF2, GFI1, IFNA1, IFNB1, IFNG, IGF1, IL1A, IL1B, IL1R1, IL1R2, IL2 , IL2RA, IL2RB, IL2RG, IL3, IL4, IL4R, IL5, IL5RA, IL6, IL6R, IL6ST, IL7, IL8, IL8RA, IL8RB, IL9, IL9R, IL10, IL10RA, IL10RB, IL11, IL11RA, IL12A, IL12B, IL12RB1 , IL12RB2, IL13, IL13RA1, IL13RA2, IL15, IL15RA, IL16, IL17, IL17R, IL18, IL18R1, IL19, IL20, KITLG, LEP, LTA, LTB, LTB4R, LTB4R2, LTBR, MIF, NPPB, PDGFB, TFX21, TDGF1, TGFA, TGFB1, TGFB1I1, TGFB2, TGFB3, TGFBI, TGFBR1, TGFBR2, TGFBR3, TH1L, TNF, TNFRSF1A, TNFRSF1B, TNFRSF7, TNFRSF8, TNFRSF9, TNFRSF11A, TNFRSF21, TNFSF4, TNFSF5, TNFSF6, TNFSF211, VEGF RNF110 (ZNF144).

asthma

Allergic asthma is characterized by the presence of eosinophilia, goblet cell metaplasia, epithelial cell changes, tracheal overreaction (AHR), Th2 and Th1 cytokine performance, and elevated serum IgE levels. Tracheitis is now widely accepted as a key factor forming the basis of the pathogenesis of asthma. It involves inflammatory cells such as T cells, B cells, eosinophils, mast cells, and macrophages and their secreting mediators, including cytokines and chemokines. ) Complex interactions. Corticosteroids are the most important anti-inflammatory treatment for asthma today, but their mechanism of action is non-specific and there are safety concerns, especially in the young patient population. Therefore, the development of more specific and targeted therapies is approved.

Animal models that can evaluate both inflammation and AHR, such as OVA-induced asthma mouse models, are known in the art and can be used to determine the ability of various DVD-Ig molecules to treat asthma. Animal models for studying asthma are disclosed in Coffman et al. , J. Exp. Med., 201 (12): 1875-1879 (2005); Lloyd et al., Adv. Immunol., 77: 263-295 (2001); Boyce et al. People, J. Exp. Med., 201 (12): 1869-1873 (2005); and Snibson et al., Clin . Exp. Allergy, 35 (2): 146-152 (2005). In addition to the routine safety assessment of these target pairs, specific tests for the extent of immunosuppression can be approved and it helps to select the best target pair (see Luster et al., Toxicology , 92 (1-3): 229 -243 (1994); Descotes, J., Develop. Biol. Standard. , 77: 99-102 (1992); Hart et al., J. Allergy Clin. Immunol. , 108 (2): 250-257 (2001) ).

One aspect of the present invention relates to a DVD-Ig molecule capable of binding to IL-1β and one or more (eg, two) targets selected from the group consisting of: IL-4, IL-5, IL-8, IL-9, IL- 13. IL-18, IL-5R (α), TNFSF4, IL-4R (α), interferon α, eosinophil chemokine, TSLP, PAR-2, PGD2 and IgE. One embodiment includes dual-specific anti-IL-1β / IL-1α DVD-Ig as a therapeutic agent beneficial for the treatment of asthma.

Rheumatoid arthritis (RA)

Rheumatoid arthritis (RA) is a systemic disease characterized by a chronic inflammatory response in the joint synovium and is associated with cartilage degradation and para-articular bone erosion. Many pro-inflammatory cytokines (including TNF, chemokines, and growth factors) appear in diseased joints. A mouse model of systemic administration of anti-TNF antibody or sTNFR fusion protein to RA has been shown to be anti-inflammatory and joint-protective. A variety of cytokines, including IL-1β, are implicated in RA. Clinical investigation of blocking TNF activity in RA patients by intravenous administration of infliximab (chimeric anti-TNF mAb) (Harriman et al., "Summary of clinical trials in rheumatoid arthritis using infliximab, an anti- "TNFalpha treatment", Ann.Rheum.Dis. , 58 (Suppl. 1): I61-I64 (1999)) is a TNF that regulates IL-6, IL-8, MCP-1 and VEGF production, immune and inflammatory cells to the joints Recruitment, angiogenesis, and reduced blood levels of matrix metalloproteinase-1 and matrix metalloproteinase-3 provide evidence. A better understanding of the inflammatory pathways in rheumatoid arthritis has led to the identification of other therapeutic targets related to rheumatoid arthritis. Such as interleukin-6 antagonists (IL-6 receptor antibody MRA, developed by Chugai, Roche (see Nishimoto et al., Arthritis Rheum., 50 (6): 1761-1769 (2004)), CTLA4Ig (Abapapp (abatacept), Genovese et al., "Abatacept for rheumatoid arthritis refractory to tumor necrosis factor alpha inhibition", N. Engl. J. Med. , 353: 1114-1123 (2005)) and anti-B cell therapy (rituximab Anti-Okamoto et al., "Rituximab for rheumatoid arthritis", N. Engl. J. Med. , 351: 1909 (2004)) promising treatments have been tested in randomized controlled trials last year. IL-Identified 1β and other cytokines (such as IL-15 and IL-18) play a role in the use of RA animal models (therapeutic antibodies HuMax-IL_15, AMG 714, see Baslund et al., Arthritis Rheum. , 52 (9): 2686- 2692 (2005)). Dual-specific antibody therapies combining anti-TNF and another mediator (the IL-1β) have great potential to enhance clinical efficacy and / or patient applicability. For example, blocking both TNF and VEGF Potentially eradicates inflammation and angiogenesis that are related to pathophysiological abnormalities of RA. It also covers the ability to block IL-1 α- and IL-1β DVD-Ig binding proteins. In addition to the routine safety assessment of these target pairs, specific tests for the extent of immunosuppression can be approved and help to select the best target pair (see Luster et al. Toxicology , 92 (1-3): 229-243 (1994); Descotes et al., Develop. Biol. Standard. , 77: 99-102 (1992); Hart et al., J. Allergy Clin. Immunol. , 108 (2): 250-257 (2001)). Preclinical animal RA models (such as a collagen-induced arthritis mouse model) can be used to evaluate whether DVD Ig molecules are suitable for treating rheumatoid arthritis. Other applicable models are also available. This technique is well known (see Brand, DD, Comp. Med. , 55: 114-122 (2005)). Based on the cross-reactivity of parental antibodies to human and mouse orthologs (e.g., othologue) (e.g., For the reactivity of human and mouse TNF, human and mouse IL-15, etc.), validation studies in mouse CIA models can be performed with "matching substitute antibody" DVD-Ig molecules. In short, DVD-Ig based on two (or more) small mouse target-specific antibodies can to the extent possible match the characteristics of a parent human or humanized antibody used in human DVD-Ig construction (similar to Affinity, similar neutralizing potency, similar half-life, etc.).

In one embodiment, the DVD-Ig of the present invention combining human IL-1β and another non-IL-1β target can also be used to treat other diseases where IL-1β functions. These diseases include (but are not limited to) SLE, multiple sclerosis (MS), sepsis, various neurological diseases and cancers (including cervical cancer, breast cancer, gastric cancer). A more exhaustive list of diseases and conditions where IL-1β functions is also provided below.

One embodiment of the present invention relates to a DVD-Ig molecule capable of binding human IL-1β and one or more targets selected from the group consisting of: IL-1α, TNFα, IL-12, TWEAK, IL-23, CXCL13, CD40, CD40L, IL-18, VEGF, VLA-4, TNFβ, CD45RB, CD200, IFN-γ, GM-CSF, FGF, C5, CD52, Sclerostin and CCR2.

Systemic lupus erythematosus (SLE)

The immunopathogenicity of SLE is the activation of multiple B cells, which results in hyperglobulinemia, autoantibody production, and immune complex formation. The main abnormality appears to be the inability of T cells to inhibit forbidden B cell pure lines due to the widespread T cell imbalance. In addition, B-cell and T-cell interactions are promoted by several cytokines (such as IL-10) and co-stimulatory molecules (such as CD40 and CD40L, B7 and CD28 and CTLA-4), which trigger a second signal. These interactions, along with the weakening of phagocytic clearance of immune complexes and apoptotic materials, maintain the immune response and cause tissue damage.

In one aspect, the DVD-Ig binding protein of the present invention is capable of binding human IL-1β and one or more of the following antigens involved in SLE: B cell targeted therapy: CD-20, CD-22, CD-19, CD28, CD4, CD80, HLA-DRA, IL10, IL2, IL4, TNFRSF5, TNFRSF6, TNFSF5, TNFSF6, BLR1, HDAC4, HDAC5, HDAC7A, HDAC9, ICOSL, IGBP1, MS4A1, RGS1, SLA2, CD81, IFNB1, IL10, TNFRSF5, TNFRSF7, TNFSF5, AICDA, BLNK, GANLAC4S-6ST, HDAC4, HDAC5, HDAC7A, HDAC9, IL10, IL11, IL4, INHA, INHBA, KLF6, TNFRSF7, CD28, CD38, CD69, CD80, CD83, CD86, DPP4, FCER2, IL2RA, TNFRSF8, TNFSF7, CD24, CD37, CD40, CD72, CD74, CD79A, CD79B, CR2, IL1R2, ITGA2, ITGA3, MS4A1, ST6GAL1, CD1C, CHST10, HLA-A, HLA-DRA and NT5E; synergistic stimulation Signal: CTLA4 or B7.1 / B7.2; Inhibition of B cell survival: BlyS, BAFF; Complement inactivation: C5; Cytokine regulation: The key principle is that the net biological response in any tissue is a pro-inflammatory or anti-inflammatory cytokine The result of a balance between regional content (see Sfikakis et al., Curr. Opin. Rheumatol. , 17: 550-557 (2005)). SLE is considered a Th-2 driven disease with a proven increase in serum IL-4, IL-6, and IL-10. Also encompassed are DVD Igs capable of binding one or more targets selected from the group consisting of IL-4, IL-6, IL-10, IFN-α and TNF-α. The combination of targets discussed herein will enhance the therapeutic efficacy of SLE, which can be tested in many preclinical models of lupus (see Peng SL, Methods Mol. Med. , 102: 227-272 (2004)). Based on the cross-reactivity of parental antibodies to human and mouse orthologs (e.g., reactivity to human and mouse CD20, human and mouse interferon alpha, etc.), validation studies in mouse lupus models can be `` matched "Substituting antibodies" from DVD-Ig molecules; in short, DVD-Ig based on two (or more) mouse-specific target antibodies can be used to the extent possible for human DVD-Ig constructs The characteristics of the human or humanized antibody are matched (similar affinity, similar neutralizing potency, similar half-life, etc.).

Multiple Sclerosis (MS)

Multiple sclerosis (MS) is a complex human autoimmune disease with a largely unknown etiology. The immunological destruction of myelin basic protein (MBP) throughout the nervous system is the main pathology of multiple sclerosis. MS is a disease with a complex pathology that involves the infiltration of CD4 + and CD8 + T cells and responses within the central nervous system. The performance of cytokines, reactive nitrogen species, and costimulatory molecules in CNS in MS has been described. The main consideration is the immunological mechanism that causes autoimmune development. In particular, the expression of antigens, the interaction of cytokines with white blood cells, and regulatory T cells that help balance / regulate other T cells such as Th1 and Th2 cells are important areas for therapeutic target identification.

IL-12 is an inflammatory cytokine that is produced by APC and promotes Th1 effector cell differentiation. IL-12 is produced in visualized lesions in patients with MS and in EAE-infected animals. Interference with the IL-12 pathway has previously been shown to be effective in preventing EAE in rodents, and has in vivo neutralization using IL-12p40 against IL-12 mAb has a beneficial effect in the myelin-induced EAE model of the common marmoset.

TWEAK is a member of the TNF family constitutively present in the central nervous system (CNS) and has pro-inflammatory, proliferative or apoptotic effects depending on the cell type. Its receptor Fn14 is expressed in the CNS by endothelial cells, reactive astrocytes and neurons. TWEAK and Fn14 mRNA expression increased in the spinal cord during experimental autoimmune encephalomyelitis (EAE). When mice are treated after the priming phase, anti-TWEAK antibody treatment in myelin oligodendritic glial cell glycoprotein (MOG) -induced EAE in C57BL / 6 mice results in reduced disease severity and Leukocyte infiltration is reduced.

One aspect of the present invention relates to a DVD Ig molecule capable of binding IL-1β and one or more (for example, two) targets selected from the group consisting of: IL-12, TWEAK, IL-23, CXCL13, CD40 , CD40L, IL-18, VEGF, VLA-4, TNF, CD45RB, CD200, IFNγ, GM-CSF, FGF, C5, CD52, osteopontin and CCR2. Examples include dual-specific anti-IL-1 β / TWEAK DVD Ig as a therapeutic agent beneficial for the treatment of MS.

Several animal models evaluating the applicability of DVD-Ig molecules for MS are known in the art (see Steinman et al., Trends Immunol. , 26 (11): 565-571 (2005); Lublin et al., Springer Semin Immunopathol. , 8 (3): 197-208 (1985); Genain et al., J. Mol. Med., 75 (3): 187-197 (1997); Tuohy et al. , J. Exp. Med. , 189 (7): 1033-1042 (1999); Owens et al., Neurol. Clin. , 13 (1): 51-73 (1995); and 't Hart et al. , J. Immunol. , 175 (7): 4761-4768 (2005)). Based on the cross-reactivity of parental antibodies to orthologs of human and animal species (eg, reactivity to human and mouse IL-1β, human and mouse TWEAK, etc.), validation studies in mouse EAE models can be "matched "Substituting antibodies" from DVD-Ig molecules; in short, DVD-Ig based on two (or more) mouse-specific target antibodies can be used to the extent possible for human DVD-Ig constructs The characteristics of the human or humanized antibody are matched (similar affinity, similar neutralizing potency, similar half-life, etc.). The same concept is applied to animal models of other non-rodent species, where a DVD-Ig of "matched alternative antibody" source is selected for expected pharmacology and possible safety studies. In addition to the routine safety assessment of these target pairs, specific tests for the extent of immunosuppression can be approved and it helps to select the best target pair (see Luster et al., Toxicology , 92 (1-3): 229 -243 (1994); Descotes et al., Develop. Biol. Standard., 77: 99-102 (1992); Jones, R., "Rovelizumab-ICOS Corp", IDrugs , 3 (4): 442-446 (2000 )).

septicemia

The pathophysiological abnormalities of sepsis are caused by outer membrane components of both Gram-negative organisms (lipopolysaccharide [LPS], lipid A, endotoxin) and Gram-positive organisms (lipoic acid, peptidoglycan). These outer membrane components are capable of binding to the CD14 receptor on the surface of monocytes. With the help of the recently described toll-like receptor, the signal is then transmitted to the cells, resulting in the eventual production of the proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1). Overwhelming inflammation and immune response are the basic characteristics of septic shock and play an important role in the pathogenesis of sepsis-induced tissue damage, multiple organ failure, and death. Cytokines, especially tumor necrosis factor (TNF) and interleukin (IL-1) have been shown to be key mediators of septic shock. These cytokines have a direct toxic effect on tissues; they also activate phospholipase A2. These and other effects result in increased concentrations of platelet activating factors, promotion of nitric oxide synthase activity, promotion of tissue infiltration of neutrophils, and promotion of neutrophil activity.

Treatment of sepsis and septic shock remains a clinical problem, and recent promising trials using inflammatory response modifiers (ie, anti-TNF, anti-MIF) against inflammatory responses have only shown limited clinical benefit. Recently, attention has shifted to therapies directed at reversing the accompanying immunosuppressive phase. Studies in experimental animals and critically ill patients have demonstrated that increased apoptosis in lymphoid organs and some parenchymal tissues results in this immunosuppression, non-responsiveness, and organ system dysfunction. During sepsis, lymphocyte apoptosis can be caused by a lack of IL-2 or by glucocorticoids, granzymes, or so-called "dead" cytokines (tumor necrosis factor alpha or Fas ligand). Triggered by release. Apoptosis occurs through the automatic activation of intracellular and / or mitochondrial Cassin, which can be affected by the pro-apoptotic and anti-apoptotic members of the Bcl-2 family. In experimental animals, treatment with apoptosis inhibitors not only prevents lymphoid cell apoptosis; it also improves results. Although clinical trials with anti-apoptotic agents are still largely remote due to technical difficulties related to their administration and tissue targeting, the inhibition of lymphocyte apoptosis represents an attractive therapeutic target for patients with sepsis. Likewise, dual specific agents that target both inflammatory mediators and apoptotic mediators may have additional benefits. One aspect of the present invention relates to a DVD Ig capable of binding to IL-1β and one or more targets related to sepsis selected from the group consisting of: TNF, IL-1, MIF, IL-6, IL-8 , IL-18, IL-12, IL-23, FasL, LPS, Toll-like receptor, TLR-4, tissue factor, MIP-2, ADORA2A, CASP1, CASP4, IL-10, IL-1B, NFKB1, PROC , TNFRSF1A, CSF3, CCR3, IL1RN, MIF, NFKB1, PTAFR, TLR2, TLR4, GPR44, HMOX1, HMG-B1, intermediate factors, IRAK1, NFKB2, SERPINA1, SERPINE1, and TREM1. The efficacy of these DVD Igs for sepsis can be evaluated in preclinical animal models known in the art (see Buras et al., Nat. Rev. Drug Discov. , 4 (10): 854-865 (2005); and Calandra Et al., Nature Med. , 6 (2): 164-170 (2000)).

Neurological disorders and neurodegenerative diseases

Neurodegenerative diseases are chronic (in this case, they are usually age-dependent) or acute (e.g., stroke, traumatic brain injury, spinal cord injury, etc.). It is characterized by progressive loss of neuronal function (neuronal cell death, demyelination), loss of activity, and loss of memory. Emerging understanding of the underlying mechanisms underlying chronic neurodegenerative diseases (such as Alzheimer's disease, AD) demonstrates complex etiology and has identified multiple factors that contribute to its formation and progression, such as age, blood glucose status, amyloid production, and Polymerization, accumulation of AGE bound to its receptor RAGE (late glycosylation end product (AGE) receptor), increased cerebral oxidative stress, decreased cerebral blood flow, neuroinflammation including release of inflammatory cytokines and chemokines, nerves Metadysfunction and Microglial Activation. As such, these chronic neurodegenerative diseases represent complex interactions between multiple cell types and mediators. The treatment strategies for these diseases are limited and mainly consist of blocking non-inflammatory processes with non-specific anti-inflammatory agents (eg, corticosteroids, COX inhibitors) or preventing the loss of neurons and / or synaptic functions with agents. These treatments have failed to stop the disease process. Recent studies have shown that more targeted therapies, such as antibodies to soluble A [beta] peptides (including A [beta] oligomeric forms) can not only help stop the disease process but also help maintain memory. These preliminary observations suggest that specific therapies that target more than one disease mediator (e.g., Aβ and pro-inflammatory cytokines (such as TNF)) can provide or even be more effective in targeting chronic neurodegenerative diseases than single disease mechanisms For example, only soluble Aβ) has been observed to have better therapeutic efficacy (see Shepherd et al., Neuropathol. Appl . Neurobiol . , 31: 503-511 (2005); Nelson, RB, Curr . Pharm . Des . , 11 : 3335-3352 (2005); Klein, WL, Neurochem. Int . , 41: 345-352 (2002); Janelsins et al., "Early correlation of microglial activation with enhanced tumor necrosis factor-alpha and monocyte chemoattractant protein-I expression specifically within the entorhinal cortex of triple transgenic Alzheimer's disease mice ", J.Neuroinflammation, 2 (23): 1-12 (2005); Soloman, B, Curr.Alzheimer.Res, 1:.. 149-163 (2004); Klyubin et al., Nature Med. , 11: 556-561 (2005); Arancio et al., EMBO J. , 23: 4096-4105 (2004); Bornemann et al . , Am.J. Pathol. , 158: 63-73 (2001); Deane et al., Nature Med, 9: 907-913 ( 2003). And Masliah et al., Neuron, 46: 857-868 (2005 )).

The DVD-Ig molecules of the present invention can bind IL-1β and one or more targets related to chronic neurodegenerative diseases such as Alzheimer's disease. These targets include, but are not limited to, any mediator (soluble or cell surface) associated with the pathogenesis of AD, such as AGE (S100 A, amphotericin), pro-inflammatory cytokines (such as IL- 1), chemokines (e.g. MCP 1), molecules that inhibit nerve regeneration (e.g. Nogo, RGM A), molecules that enhance neurite growth (neurotrophic factors), and molecules that mediate transport at blood-brain barriers ( (Eg, transferrin receptor, insulin receptor or RACE). The efficacy of DVD-Ig molecules can be verified in preclinical animal models, such as over-expression of amyloid precursor protein Or RAGE transgenic mice with Alzheimer's disease-like symptoms. In addition, DVD-Ig molecules can be constructed and tested for efficacy in animal models and the best therapeutic DVD-Ig can be selected for testing in human patients. DVD-Ig molecules can also be used to treat other neurodegenerative diseases, such as Parkinson's disease. Alpha-synuclein is involved in the pathology of Parkinson's disease. DVD-Ig capable of targeting IL-1β and LINGO-1, α-synuclein and / or inflammatory mediators such as TNF, IL-1, MCP-1 can prove to be effective treatments for Parkinson's disease and Covered by the present invention.

Neuron regeneration and spinal cord injury

Despite increased understanding of pathological mechanisms, spinal cord injury (SCI) remains a devastating condition and represents a medical indication characterized by a high medical need. Most spinal cord injuries are contusions or crush injuries, and the primary injury is usually followed by a secondary injury mechanism (inflammatory mediators, such as cytokines and chemokines), which worsens the initial injury and causes the lesion area to expand significantly. More than 10 times. These primary and secondary mechanisms in SCI are very similar to those caused by other methods, such as stroke, in brain injury. There is no satisfactory treatment and high-dose rapid injection of methylprednisolone (MP) is the only therapy used within a narrow time window of 8 hours after injury. However, this treatment is only intended to prevent secondary damage without causing any significant functional recovery. It has been heavily criticized for its lack of clear efficacy and severe adverse effects such as immunosuppression, subsequent infections, and severe histopathological muscle changes. Other drugs, biologics, or small molecules that stimulate endogenous regeneration potential have not been approved, but promising therapeutic principles and drug candidates have shown efficacy in animal models of SCI in recent years. To a large extent, the lack of functional recovery in human SCI is caused by factors that inhibit neurite growth at the site of the lesion, in scar tissue, in myelin, and on damage-related cells. These factors are myelin-related proteins NogoA, OMgp and MAG, RGM A, scar-related CSPG (chondroitin sulfate proteoglycan), and inhibitors of reactive astrocytes (semaphorin and ephrin ). However, not only growth inhibitory molecules but also neurite growth stimulating factors such as neurotrophic factors, laminin, L1, and others were found at the lesion site. This aggregate of neurite growth inhibition and growth promoting molecules can explain that blocking a single factor (such as NogoA or RGM A) leads to significant functional recovery in rodent SCI models, as reduced inhibition effects can shift the balance from growth inhibition to growth promotion . However, the recovery observed with regard to blocking a single neurite outgrowth inhibitory molecule is incomplete. For faster and more significant recovery, block two neurite outgrowth inhibitory molecules (such as Nogo and RGM A) or block neurite outgrowth inhibitory molecules and enhance neurite outgrowth enhancing molecules (such as Nogo and neurotrophic factors) ), Or blocking neurite outgrowth inhibitory molecules (such as Nogo) and pro-inflammatory molecules (such as TNF) may be needed (see McGee et al., Trends Neurosci. , 26: 193-198 (2003); Domeniconi Et al., J. Neurol. Sci. , 233: 43-47 (2005); Makwana et al., FEBS J. , 272: 2628-2638 (2005); Dickson, BJ, Science , 298: 1959-1964 (2002) Teng et al., J. Neurosci. Res. , 79: 273-278 (2005); Karnezis et al., Nature Neurosci. , 7: 736 (2004); Xu et al. , J. Neurochem. , 91: 1018-1023 (2004)).

In one aspect, DVD-Ig that binds human IL-1β can also bind one or two target pairs such as: NgR and RGM A; NogoA and RGM A; MAG and RGM A; OMgp and RGM A; RGM A And RGM B; CSPGs and RGM A; aggrecan, mid-term factor, neurocan, multifunctional proteoglycan (versican), phosphacan, Te38 and TNF-α Provide a combination of Aβ globulomer-specific antibodies and antibodies that promote dendritic & axonal branching. Dendritic lesions are very early signs of AD and NOGO A is known to restrict dendritic growth. This type of ab can be combined with SCI-candidate (myelin-protein) Ab. Other DVD-Ig targets may include any combination of the following: NgR-p75, NgR-Troy, NgR-Nogo66 (Nogo), NgR-Lingo, Lingo-Troy, Lingo-p75, MAG, or OMgp. In addition, targets can include any mediator (soluble or cell surface) associated with the inhibition of neurites, such as Nogo, OMpg, MAG, RGM A, signaling proteins, ephrin, soluble Aβ, pro-inflammatory cytokines (Such as IL-1), chemokines (such as MIP 1a), molecules that inhibit neural regeneration. The efficacy of anti-nogo / anti-RGM A or DVD-Ig-like molecules can be verified in preclinical animal models of spinal cord injury. In addition, these DVD-Ig molecules can be constructed and tested for efficacy in animal models and the best therapeutic DVD-Ig can be selected for testing in human patients. In addition, DVD-Ig molecules can be constructed that target two different ligand binding sites on a single receptor, such as the Nogo receptor that binds the three ligands Nogo, OMGp, and MAG, and binds Aβ And RAGE of S100 A. In addition, neurite outgrowth inhibitors (such as nogo and nogo receptors) also play a role in preventing nerve regeneration in immune diseases such as multiple sclerosis. Inhibition of nogo-nogo receptor interactions in animal models of multiple sclerosis has been shown to enhance recovery. Therefore, a DVD-Ig molecule that can block the function of an immune mediator (e.g., a cytokine, such as IL-12) and an neurite outgrowth inhibitory molecule (e.g., Nogo or RGM) can provide a greater advantage than blocking immune or Epistatic molecules are faster and more potent.

In general, antibodies do not cross the blood brain barrier (BBB) in an effective and relevant manner. However, in the case of certain neurological diseases such as stroke, traumatic brain injury, multiple sclerosis, etc., the BBB may be damaged and allow increased DVD-Ig and antibodies to penetrate into the brain. In other neurological conditions where BBB leaks do not occur, targeted endogenous delivery systems can be used, including carrier-mediated transporters such as glucose and amino acid carriers, and receptor-mediated transcytosis, Mediates cellular structures / receptors at the vascular endothelium of the BBB, thus enabling DVD-Ig delivery across the BBB. The structures at the BBB that enable this delivery include, but are not limited to, the insulin receptor, transferrin receptor, LRP, and RAGE. In addition, the strategy enables DVD-Ig to be used as a shuttle to deliver potential drugs to the CNS, including low molecular weight drugs, nanoparticle and nucleic acid (Coloma et al., Pharm.Res. , 17 (3): 266-274 (2000); Boado et al., Bioconjug. Chem . , 18 (2): 447-455 (2007)).

Carcinogenic condition

Monoclonal antibody therapy has emerged as an important therapeutic modality for cancer (von Mehren et al., Ann. Rev. Med . , 54: 343-369 (2003)). Antibodies can exert antitumor effects by inducing apoptosis, redirecting cytotoxicity, interfering with ligand-receptor interactions, or preventing the expression of proteins that are critical for neoplastic phenotypes. In addition, antibodies can target components of the tumor microenvironment, thereby disrupting vital structures such as the formation of tumor-associated vascular structures. Antibodies can also target receptors whose ligands are growth factors, such as the epidermal growth factor receptor. Therefore, antibodies can inhibit the binding of natural ligands that stimulate cell growth to the targeted tumor cells. Alternatively, antibodies can induce anti-idiotypic networks, complement-mediated cytotoxicity, or antibody-dependent cytotoxicity (ADCC). Compared with monospecific therapy, the use of dual specific antibodies that target two independent tumor mediators may yield additional benefits.

In another embodiment, the DVD-Ig combined with human IL-1β of the present invention may also be able to bind another target related to carcinogenic diseases, including (but not limited to): IGFR, IGF, VGFR1, PDGFRb, PDGFRa , IGF1,2, ERB3, CDCP, 1BSG2, ErbB3, CD52, CD20, CD19, CD3, CD4, CD8, BMP6, IL12A, IL1A, IL1B, IL2, IL24, INHA, TNF, TNFSF10, BMP6, EGF, FGF1, FGF10 , FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GRP, IGF1, IGF2, IL12A, ILA , IL1B, IL2, INHA, TGFA, TGFB1, TGFB2, TGFB3, VEGF, CDK2, FGF10, FGF18, FGF2, FGF4, FGF7, IGF1R, IL2, BCL2, CD164, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2C, CDKN2C , GNRH1, IGFBP6, IL1A, IL1B, ODZ1, PAWR, PLG, TGFB1I1, AR, BRCA1, CDK3, CDK4, CDK5, CDK6, CDK7, CDK9, E2F1, EGFR, ENO1, ERBB2, ESR1, ESR2, IGFBP3, IGFBP6, IL2 , INSL4, MYC, NOX5, NR6A1, PAP, PCNA, PRKCQ, PRKD1, PRL, TP53, FGF22, FG F23, FGF9, IGFBP3, IL2, INHA, KLK6, TP53, CHGB, GNRH1, IGF1, IGF2, INHA, INSL3, INSL4, PRL, KLK6, SHBG, NR1D1, NR1H3, NR1I3, NR2F6, NR4A3, ESR1, ESR2, NR0B1, NR0B2, NR1D2, NR1H2, NR1H4, NR1I2, NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR3C1, NR3C1, NR3C1 NR4A2, NR5A1, NR5A2, NR6A1, PGR, RARB, FGF1, FGF2, FGF6, KLK3, KRT1, APOC1, BRCA1, CHGA, CHGB, CLU, COL1A1, COL6A1, EGF, ERBB2, ERK8, FGF1, FGF10, FGF11, FGF13, FGF14, FGF16, FGF17, FGF18, FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GNRH1, IGF1, IGF2, IGFBP3, IGFBP6, IL12A, IL1A, IL1B, IL2, IL24, INHA, INSL3, INSL4, KLK10, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, MMP2, MMP9, MSMB, NTN4, ODZ1, PAP, PLAU, PRL, PSAP, SERPINA3, SHBG, TGFA, TIMP3, CD44, CDH1, CDH10, CDH19, CDH20, CDH7, CDH9, CDH1, CDH10, CDH13, CDH18, CDH19, CDH20, CDH7, CDH8, CDH9, ROBO2, CD44, ILK, ITGA1, APC, CD164, COL6A1 MTSS1, PAP, TGFB1I1, AGR2, AIG1, AKAP1, AKAP2, CANT1 CAV1, CDH12, CLDN3, CLN3, CYB5, CYC1, DAB2IP, DES, DNCL1, ELAC2, ENO2, ENO3, FASN, FLJ12584, FLJ25530, GAGEB1, GAGEC1, GGT1, GSTP1, HIP1, HUMCYT2A, IL29, K6HF, KAI1, KRT2A, IL29 MIB1, PART1, PATE, PCA3, PIAS2, PIK3CG, PPID, PR1, PSCA, SLC2A2, SLC33A1, SLC43A1, STEAP, STEAP2, TPM1, TPM2, TRPC6, ANGPT1, ANGPT2, ANPEP, ECGF1, EREG, FGF1, FGF2, FIGF, FIGF FLT1, JAG1, KDR, LAMA5, NRP1, NRP2, PGF, PLXDC1, STAB1, VEGF, VEGFC, ANGPTL3, BAI1, COL4A3, IL8, LAMA5, NRP1, NRP2, STAB1, ANGPTL4, PECAM1, PF4, PROK2, SERPINF1, TNFAIP2, CCL11, CCL2, CXCL1, CXCL10, CXCL3, CXCL5, CXCL6, CXCL9, IFNA1, IFNB1, IFNG, IL1B, IL6, MDK, EDG1, EFNA1, EFNA3, EFNB2, EGF, EPHB4, FGFR3, HGF, IGF1, ITGB3, PDGFA, TEK, TGFA, TGFB1, TGFB2, TGFBR1, CCL2, CDH5, COL18A1, EDG1, ENG, ITGAV, ITGB3, THBS1, THBS2, BAD, BAG1, BCL2, CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CDH1 (E-cadherin), CDKN1B (p27Kip1), CDKN2A (p16INK4a), COL6A1, CTNNB1 (b-soparin), CTSB (Cathepsin B), ERBB2 (Her-2), ESR1, ESR2, F3 (TF), FOSL1 (FRA-1), GATA3, GSN (peptin), IGFBP2, IL2RA, IL6, IL6R, IL6ST (sugar Protein 130), ITGA6 (a6 integrin), JUN, KLK5, KRT19, MAP2K7 (c-Jun), MKI67 (Ki-67), NGFB (NGF), NGFR, NME1 (NM23A), PGR, PLAU (uPA), PTEN, SERPINB5 (maspin), SERPINE1 (PAI-1), TGFA, THBS1 (thrombin-1), TIE (Tie-1), TNFRSF6 (Fas), TNFSF6 (FasL), TOP2A ( Topoisomerase Iia), TP53 AZGP1 (zinc-a-glycoprotein), BPAG1 (reticulin), CDKN1A (p21Wap1 / Cip1), CLDN7 (close junction protein-7), CLU (agglutinin), ERBB2 (Her-2), FGF1, FLRT1 (fiber (Catenin), GABRP (GABAa), GNAS1, ID2, ITGA6 (a6 integrin), ITGB4 (b 4 integrin), KLF5 (GC Box BP), KRT19 (keratin 19), KRTHB6 (hair-specific type II keratin ), MACMARCKS, MT3 (metal sulfur-binding protein-III), MUC1 (mucin), PTGS2 (COX-2), RAC2 (p21Rac2), S100A2, SCGB1D2 (lipophilin B), SCGB2A1 (mammoglobulin 2), SCGB2A2 (mammoglobulin 1), SPRR1B (Spr1), THBS1, THBS2, THBS4 and TNFAIP2 (B94), RON, c-Met, CD64, DLL4, PLGF, CTLA4, phosphorus Fatty Serine, ROBO4, CD80, CD22, CD40, CD23, CD28, CD55, CD38, CD70, CD74, CD30, CD138, CD56, CD33, CD2, CD137, DR4, DR5, RANKL, VEGFR2, PDGFR, VEGFR1, MTSP1, MSP, EPHB2, EPHA1, EPHA2, EpCAM, PGE2, NKG2D, LPA, SIP, APRIL, BCMA, MAPG, FLT3, PDGFRα, PDGFRβ, ROR1, PSMA, PSCA, SCD1, and CD59.

D. Pharmaceutical composition

The invention also provides a pharmaceutical composition comprising an antibody of the invention (including a DVD-Ig as described herein) or an antigen-binding portion thereof, and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising an antibody of the invention is used for, but not limited to, diagnosing, detecting, or monitoring a disorder, for preventing, treating, controlling, or ameliorating a disorder or one or more symptoms thereof, and / or for research. In a particular embodiment, the composition comprises one or more antibodies of the invention. In another embodiment, the pharmaceutical composition comprises one or more antibodies of the invention and one or more prophylactic or therapeutic agents other than the antibodies of the invention for treating a condition in which IL-1β activity is detrimental. In one embodiment, the prophylactic or therapeutic agent is known to be suitable or has been used or is currently used to prevent, treat, control or ameliorate a condition or one or more symptoms thereof. According to these embodiments, the composition may further include a carrier, diluent, or excipient.

The antibodies and antibody portions of the present invention can be incorporated into a pharmaceutical composition suitable for administration to an individual. Generally, a pharmaceutical composition comprises an antibody or antibody portion of the invention and a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" as used herein includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible . Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further contain minor amounts of auxiliary substances that enhance the shelf life or effectiveness of the antibody or antibody portion, Such as wetting or emulsifying agents, preservatives or buffering agents.

Various delivery systems are known and can be used to administer one or more antibodies of the invention or one or more antibodies of the invention in combination with prophylactic or therapeutic agents suitable for preventing, controlling, treating or ameliorating a disorder or one or more symptoms thereof , Such as encapsulated in liposomes, microparticles, microcapsules; recombinant cells capable of expressing antibodies or antibody fragments; receptor-mediated internal drinking effects (see, eg, Wu and Wu, J. Biol. Chem. 262: 4429-4432 (1987)); constructing nucleic acids, etc. as part of a retrovirus or other vector. Methods of administering the prophylactic or therapeutic agents of the present invention include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural administration, intratumoral administration And mucosal administration (eg, intranasal and oral routes). Alternatively, pulmonary administration may be used, for example, by using an inhaler or nebulizer, and formulated with an atomizer. See, for example, U.S. Patent Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, and 5,290,540; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903, each of which is incorporated herein by reference in its entirety. In one embodiment, the antibody or antibody portion, combination therapy, or composition of the invention is administered using Alkermes AIR® pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Massachusetts). In a specific embodiment, the prophylactic or therapeutic agent of the present invention is administered intramuscularly, intravenously, intratumorally, intraorally, intranasally, pulmonary or subcutaneously. Prophylactic or therapeutic agents can be administered by any suitable route, such as by infusion or rapid injection, absorbed through the epithelial or mucosal skin lining (such as oral mucosa, rectum, and intestinal mucosa, etc.), and can be combined with other bioactive agents Vote for. Administration can be systemic or local.

In one embodiment, the antibody-coupled carbon nanotube (CNT) specifically binds to tumor cells in vitro, and then its highly specific resection by near-infrared (NIR) light can be used to target tumor cells. For example, biotin-labeled polar lipids can be used to prepare stable, biocompatible, non-cytotoxic CNT dispersions that are subsequently linked to one or two different neutralites directed against one or more tumor antigens, such as CD22 ) Avidin-derived DVD-Ig (Chakravarty et al., Proc. Natl. Acad. Sci. USA , 105: 8697-8702 (2008)).

In a particular embodiment, it may be necessary to locally administer the prophylactic or therapeutic agent of the present invention to areas in need of treatment; this can be achieved by, for example, but not limited to, a local infusion, injection, or by means of an implant, The implant is a porous or non-porous material, including a membrane and a matrix, such as a sialastic membrane, a polymer, a fibrous matrix (such as Tissuel®), or a collagen matrix. In one embodiment, an effective amount of one or more antibody antagonists of the invention is administered topically to an affected area of an individual to prevent, treat, control and / or ameliorate a disorder or its symptoms. In another embodiment, an effective amount of one or more antibodies of the invention and an effective amount of one or more therapies (e.g., one or more prophylactic or therapeutic agents) other than the antibodies of the invention are administered locally to the affected area of the subject To prevent, treat, control and / or ameliorate a condition or one or more symptoms thereof.

In another embodiment, a controlled release or sustained release system can be used to deliver a prophylactic or therapeutic agent. In one embodiment, controlled release or sustained release can be achieved using a pump (see Langer, supra; Sefton, MV, CRC Crit. Rev. Biomed . Eng . , 14: 201-240 (1987); Buchwald et al., Surgery , 88: 507-516 (1980); Saudek et al., N. Engl. J. Med. , 321: 574-579 (1989)). In another embodiment, polymeric materials can be used to achieve controlled release or sustained release of the invention therapy (see, for example, Goodson, JM, Chapter 6, Medical Applications of Controlled Release, Volume II, Applications and Evaluation , (Langer and Wise (Ed.) (CRC Press, Inc., Boca Raton, 1984) pp. 115-138; Langer and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. Phys . , C23 (1): 61-126 (1983) ; See also Levy et al., Science , 228: 190-192 (1985); During et al. , Ann. Neurol. , 25: 351-356 (1989); Howard et al., J. Neurosurg . , 71: 105-112 (1989); US Patent No. 5,679,377; US Patent No. 5,916,597; US Patent No. 5,912,015; US Patent No. 5,989,463; US Patent No. 5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No. WO 99/20253). Examples of polymers used in sustained release formulations include, but are not limited to, poly (2-hydroxyethyl methacrylate), poly (methyl methacrylate), poly (acrylic acid), poly (ethylene-co-acetic acid) Vinyl ester), poly (methacrylic acid), polyglycolide (PLG), polyanhydride, poly (N-vinylpyrrolidone), poly (vinyl alcohol), polyacrylamide, poly (ethylene glycol), Polylactide (PLA), poly (lactide-co-glycolide) (PLGA), and polyorthoesters. In an exemplary embodiment, the polymer used in the sustained-release formulation is inert, contains no leachable impurities, is stable during storage, is sterile, and is biodegradable. In another embodiment, a controlled release or sustained release system can be placed adjacent to a prophylactic or therapeutic target, thereby requiring only a portion of the systemic dose (see, eg, Goodson, Medical Applications of Controlled Release , supra, vol. 2, 115-138 (1984)).

Controlled release systems are discussed in the review by Langer ( Science , 249: 1527-1533 (1990)). Any technique known to those skilled in the art can be used to produce sustained release formulations comprising one or more therapeutic agents of the invention. See, for example, U.S. Patent No. 4,526,938; PCT Publication No. WO 91/05548; PCT Publication No. WO 96/20698; Ning et al., "Intratumoral radioimmunotherapy of a human colon cancer xenograft using a sustained-release gel", Radiotherapy Oncol., 39: 179-189 (1996); Song et al. "Antibody Mediated Lung Targeting of Long-Circulating Emulsions", PDA J. Pharm. Sci. Technol . , 50: 372-377 (1996); Cleek et al. "Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application", Proceed.Int'l.Symp.Control.Rel.Bioact.Mater. , 24: 853-854 (1997); and Lam et al., "Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery ", Proceed.Int'l.Symp.Control Rel.Bioact.Mater. , 24: 759-760 (1997), each of which is incorporated herein by reference in its entirety.

In a specific embodiment where the composition of the present invention is a nucleic acid encoding a prophylactic or therapeutic agent, the nucleic acid can be administered in vivo by constructing it as part of a suitable nucleic acid expression vector and administering it so that it becomes an intracellular nucleic acid. The nucleic acid promotes the performance of the encoded preventive or therapeutic agent, for example, by using a retroviral vector (see US Patent No. 4,980,286), or by direct injection, or by bombardment with particles (Eg gene gun; Biolistic®, DuPont), or coated with a lipid or cell surface receptor or transfection agent, or by linking it with a homeobox-like peptide known to enter the nucleus Administration (see, eg, Joliot et al., Proc. Natl. Acad. Sci. USA , 88: 1864-1868 (1991)). Alternatively, the nucleic acid can be introduced into a cell and incorporated into the host cell DNA for expression by homologous recombination.

The pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include, but are not limited to, parenteral (e.g. intravenous, intradermal, subcutaneous), oral, intranasal (e.g. inhalation), transdermal (e.g. topical), transmucosal and rectal administration. In a specific embodiment, the composition is formulated into a pharmaceutical composition suitable for intravenous, subcutaneous, intramuscular, oral, intranasal or topical administration to humans according to conventional procedures. Generally, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocamne to reduce pain at the injection site.

If the composition of the present invention is to be administered topically, the composition can be formulated into ointments, creams, transdermal patches, lotions, gels, shampoos, sprays, aerosols, solutions, and emulsions. Form or other forms known to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms , 19th edition, Mack Pub. Co., Easton, Pa. (1995). For non-sprayable topical dosage forms, a viscous to semi-solid or solid form containing a carrier or one or more excipients compatible with topical application and preferably having a dynamic viscosity greater than water is generally employed. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, elixirs, ointments, and the like, and sterilize or, if necessary, affect properties such as osmotic pressure. Adjuvants (such as preservatives, stabilizers, wetting agents, buffers, or salts) are mixed. Other suitable topical dosage forms include sprayable aerosol formulations, where the active ingredient preferably combined with a solid or liquid inert carrier is mixed with a pressurized volatile substance (e.g., a gaseous propellant such as FREON®) or packaged in an extrusion In the bottle. If necessary, a moisturizing agent or a humectant may be added to the pharmaceutical composition and the dosage form. Examples of these other ingredients are well known in the art.

If the method of the present invention comprises administering the composition intranasally, the composition can be formulated in the form of an aerosol, a spray, a mist or a drop. In particular, the preventive or therapeutic agent used in accordance with the present invention is preferably derived from a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas Aerosol sprays for compression packaging or sprayers are delivered in the form of a spray. In the case of pressurized aerosols, the dosage unit can be determined by providing a valve to deliver the metered amount. Capsules and kits (consisting of, for example, gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mixture of the compound with a suitable powder base such as lactose or starch.

If the method of the present invention comprises oral administration, the composition can be formulated into oral lozenges, capsules, cachets, gelcaps, solutions, suspensions and the like. Lozenges or capsules may be prepared in a conventional manner using pharmaceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethyl) Cellulose); fillers (such as lactose, microcrystalline cellulose or dibasic calcium phosphate); lubricants (such as magnesium stearate, talc or silicon dioxide); disintegrants (such as potato starch or sodium starch glycolate); Or wetting agent (such as sodium lauryl sulfate). Lozenges can be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, but are not limited to, solutions, syrups or suspensions, or they may be in the form of a dried product which is reconstituted with water or other suitable vehicle before use. These liquid preparations may be prepared in a conventional manner with pharmaceutically acceptable additives such as suspending agents (such as sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifiers (such as lecithin or Gum Arabic); non-aqueous vehicle (e.g. apricot Kernel oil, oily ester, ethanol or fractionated vegetable oil); and preservatives (such as methyl paraben or propyl paraben or sorbic acid). Where appropriate, these preparations may also contain buffer salts, flavoring agents, coloring agents, and sweetening agents. Formulations for oral administration should preferably be formulated for slow-, controlled-, or sustained-release prophylactic or therapeutic agents.

The method of the invention may include, for example, pulmonary administration of a composition formulated with an atomizer by using an inhaler or a nebulizer. See, for example, U.S. Patent Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, and 5,290,540; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903, each of which is incorporated herein by reference in its entirety. In a specific embodiment, the antibody, combination therapy, and / or composition of the invention is administered using Alkermes AIR® pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Massachusetts).

The method of the invention may include administering a composition formulated for parenteral administration by injection (eg, by bolus injection or continuous infusion). Formulations for injection may be presented in unit dosage forms (eg, in ampoules or multi-dose containers) with preservatives. The composition may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle, and may contain formulating agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be in the form of a powder that is reconstituted with a suitable vehicle, such as sterile pyrogen-free water, before use.

The method of the present invention may further comprise administering a composition formulated into a tank formulation. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, it can be formulated with suitable polymeric or hydrophobic materials (e.g. in the form of emulsions in acceptable oils) or ion exchange resins, or in the form of sparingly soluble derivatives (e.g. in the form of sparingly soluble salts) combination.

The method of the invention encompasses administration of a composition formulated into a neutral or salt form. Pharmaceutically acceptable salts include salts formed with anions, such as derived from hydrochloric acid, phosphoric acid, acetic acid, Salts of oxalic acid, tartaric acid, etc .; and salts formed with cations, such as derived from sodium, potassium, ammonium, calcium, iron hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histamine, proca Wait for the salt.

Generally, the ingredients of the composition are provided individually or in unit dosage forms mixed together (for example, in the form of a dry lyophilized powder or anhydrous concentrate) in a closed container, such as an ampoule or sachet, indicating the active dose. When the mode of administration is infusion, the composition can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the administration mode is injection, an ampoule of sterilized water or saline for injection can be provided to mix the ingredients before administration.

The present invention also particularly provides for encapsulating one or more of the preventive or therapeutic agents or pharmaceutical compositions of the present invention in a closed container (such as an ampoule or sachet) indicating the dose of the drug. In one embodiment, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions of the present invention are provided in a closed container in the form of a dry sterilized lyophilized powder or an anhydrous concentrate and are reconstituted (e.g., water or saline) to a suitable The concentration administered to the individual. One or more prophylactic or therapeutic agents or pharmaceutical compositions of the present invention are preferably in a unit dose of at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 75 mg or at least 100 mg, Provided in a dry, sterile lyophilized powder in a closed container. The lyophilized preventive or therapeutic agent or pharmaceutical composition of the present invention should be stored in its original container between 2 ° C and 8 ° C, and the preventive or therapeutic agent or pharmaceutical composition of the present invention should be 1 week after recovery Within, preferably within 5 days, within 72 hours, within 48 hours, within 24 hours, within 12 hours, within 6 hours, within 5 hours, within 3 hours, or within 1 hour. In an alternative embodiment, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions of the present invention are provided in a liquid form in a closed container indicating the amount and concentration of the agent. The administered composition in liquid form is preferably at least 0.25 mg / ml, more preferably at least 0.5 mg / ml, at least 1 mg / ml, at least 2.5 mg / ml, at least 5 mg / ml, at least 8 mg / ml, at least 10 mg / ml , At least 15 mg / kg, at least 25 mg / ml, at least 50 mg / ml, at least 75 mg / ml, or at least 100 mg / ml are provided in a closed container. The liquid form should be stored in its original container between 2 ° C and 8 ° C.

The antibodies and antibody portions of the present invention can be incorporated into pharmaceutical compositions suitable for parenteral administration. The antibody or antibody portion is preferably made into an injectable solution containing 0.1-250 mg / ml antibody. The injectable solution may consist of liquid or lyophilized dosage forms in hard or amber vials, ampoules or pre-filled syringes. The buffer may be L-histidine (1-50 mM), preferably 5-10 mM, and a pH value of 5.0 to 7.0 (optimal pH value 6.0). Other suitable buffering agents include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate, or potassium phosphate. Sodium chloride at a concentration of 0-300 mM (optimally 150 mM for liquid dosage forms) can be used to alter the toxicity of the solution. For lyophilized dosage forms, cryoprotectants can be included, mainly 0-10% sucrose (optimally 0.5-1.0%). Other suitable cryoprotectants include trehalose and lactose. For lyophilized dosage forms, a bulking agent may be included, mainly 1-10% mannitol (optimally 2-4%). Stabilizers can be used in both liquid and lyophilized formulations, mainly 1-50 mM L-methionine (optimally 5-10 mM). Other suitable accumulating agents include glycine and arginine, which can be included in the form of 0-0.05% polysorbate-80 (optimally 0.005-0.01%). Other surfactants include, but are not limited to, polysorbate 20 and BRIJ surfactants. A pharmaceutical composition comprising an antibody or antibody portion of the present invention in the form of an injectable solution for parenteral administration may further comprise an agent that can be used as an adjuvant, such as for increasing the therapeutic protein (e.g., antibody) Absorbed or dispersed medicament. A particularly suitable adjuvant is hyaluronidase (such as Hylenex® recombinant human hyaluronidase). Adding hyaluronidase to the injectable solution improves the human bioavailability after parenteral administration, especially after subcutaneous administration. It also allows for a larger injection site volume (ie, greater than 1 ml) with less pain and discomfort, and minimizes the incidence of injection site reactions (see PCT Publication No. WO 2004/078140 and US Publication No. 2006 / 104968).

The composition of the invention may take a variety of forms. Such forms include, for example, liquid, semi-solid, and solid dosage forms such as liquid solutions (e.g., injectable solutions and infusible solutions), dispersions or suspensions, lozenges, pills, powders, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, such as combinations similar to those used to passively immunize humans with other antibodies Thing. The preferred mode of administration is parenteral (eg, intravenous, subcutaneous, intraperitoneal, intramuscular). In an exemplary embodiment, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibodies are administered by intramuscular or subcutaneous injection.

Therapeutic compositions must generally be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for higher drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., the antibody or antibody portion) in the required amount with one or a combination of the ingredients listed above in a suitable solvent, followed by filtration and sterilization if necessary. Dispersions are generally prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of a sterile lyophilized powder for the preparation of a sterile injectable solution, the preferred method of preparation is vacuum drying and spray drying, which results in a powder of the active ingredient plus any other desired ingredients from a previously sterile filtered solution. Proper fluidity of the solution can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. Prolonged absorption of the composition can be achieved by including delayed absorption agents such as monostearate and gelatin in the injectable composition.

The binding protein of the present invention can be administered by a variety of methods known in the art, but for many therapeutic applications, the preferred route / mode of administration is subcutaneous injection, intravenous injection or infusion. As understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired result. In certain embodiments, the active compound may be prepared with a vehicle that protects the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods of preparing such formulations are patented or generally known to those skilled in the art. See, for example, see, for example , Sustained and Controlled Release Drug Delivery Systems , (ed. JR Robinson) (Marcel Dekker, Inc., New York, 1978).

In certain embodiments, an antibody or antibody portion of the invention can be, for example, an inert diluent Or it can be administered orally with an assimilated edible carrier. The compounds (with other ingredients if necessary) can also be enclosed in hard or soft shell gelatin capsules, compressed into lozenges, or incorporated directly into the diet of the individual. For oral therapeutic administration, the compounds may be combined with excipients and used in the form of ingestible lozenges, buccal tablets, dragees, capsules, elixirs, suspensions, syrups, wafers and the like . In order to administer the compound of the present invention by means other than enteral administration, it may be necessary to coat the compound with a substance to prevent inactivation of the compound, or to co-administer the compound with a substance to prevent inactivation of the compound.

Supplementary active compounds can also be incorporated into the composition. In certain embodiments, an antibody or antibody portion of the invention is co-formulated and / or co-administered with one or more other therapeutic agents suitable for treating conditions in which IL-1β activity is detrimental. For example, the anti-human IL-1β antibody or antibody portion of the present invention can be co-formulated and / or co-administered with one or more other antibodies that bind other targets (such as antibodies that bind other cytokines or cell surface molecules). versus. In addition, one or more antibodies of the present invention can be used in combination with two or more of the foregoing therapeutic agents. These combination therapies should preferably utilize lower doses of the administered therapeutic agent, thereby avoiding possible toxicity or complications associated with various monotherapy.

In certain embodiments, an antibody or fragment thereof directed to IL-Ιβ is linked to a half-life extending vehicle known in the art. Such vehicles include, but are not limited to, the Fc domain, polyethylene glycol, and polydextrose. Such vehicles are described, for example, in US Application No. 09 / 428,082 (now US Patent No. 6,660,843), which is incorporated herein by reference for any purpose.

In a specific embodiment, a nucleic acid sequence comprising a nucleotide sequence encoding an antibody of the invention or another preventive or therapeutic agent of the invention is administered to treat, prevent, control or ameliorate a condition or one or more thereof via gene therapy symptom. Gene therapy refers to a therapy performed by administering an expressed or expressible nucleic acid to an individual. In this embodiment of the invention, the nucleic acid produces an antibody or prophylactic or therapeutic agent of the invention that it encodes to mediate a prophylactic or therapeutic effect.

Any method available in the art for gene therapy can be used in accordance with the present invention. For a general review of methods of gene therapy, see Goldspiel et al., Clinical Pharm. , 12: 488-505 (1993); Wu et al., "Delivery systems for gene therapy", Biotherapy , 3: 87-95 (1991); Tolstoshev, P., Ann. Rev. Pharmacol. Toxicol . , 32: 573-596 (1993); Mulligan, RC, Science , 260: 926-932 (1993); and Morgan and Anderson, "Human Gene Therapy", Ann Rev. Biochem. , 62: 191-217 (1993); Robinson, C., Trends Biotechnol. , 11: 155 (1993). Methods commonly known in the art for use of recombinant DNA technology are described in Ausubel et al. (Eds.), Current Protocols in Molecular Biology , John Wiley & Sons, New York (1993); and Kriegler, Gene Transfer and Expression, Laboratory Manual Stockton Press, New York (1990). A detailed description of various methods of gene therapy is disclosed in US Publication No. 2005/0042664 A1, which is incorporated herein by reference.

Members of the IL-1 family (IL-1β and IL-1α) play a key role in the pathology associated with a variety of diseases involving immune and inflammatory elements. The IL-1 binding proteins described herein can be administered to individuals to treat these conditions. In one embodiment, conditions that can be treated by a method of the invention comprising administering to a subject an IL-1 binding protein described herein include, but are not limited to, diabetes; uveitis; neuralgia; osteoarthritis pain Inflammatory pain; rheumatoid arthritis; osteoarthritis; juvenile chronic arthritis; septic arthritis; Lyme arthritis; psoriasis arthritis; reactive arthritis; spinal arthropathy; systemic lupus erythematosus ( SLE); Crohn's disease; ulcerative colitis; inflammatory bowel disease; autoimmune diabetes; insulin-dependent diabetes; thyroiditis; asthma; allergic disease; psoriasis; dermatitis; scleroderma; graft-versus-host Disease; organ transplant rejection; acute immune disease related to organ transplant; chronic immune disease related to organ transplant; sarcomatoid disease; atherosclerosis; disseminated intravascular coagulation (DIC); Kawasaki's disease; Gray disease Renal disease syndrome; Chronic fatigue syndrome; Wegener's granulomatosis; Heng-Schienlai purpura; Renal microangitis; Chronic active hepatitis; Autoimmune Grapes meningitis; septic shock; poisoning Septic syndrome; sepsis syndrome; cachexia; infectious disease; parasitic disease; acute transverse myelitis; Huntington's disease; Parkinson's disease; Alzheimer's disease; stroke; primary biliary cirrhosis Hemolytic anemia; Malignant disease; Heart failure; Myocardial infarction; Addison's disease; Occasional type 1 polyglandular hyposecretion; Type II polyglandular hyposecretion (Schmidt's syndrome); Acute respiratory distress Syndrome (ARDS); hair loss; patchy hair loss; sero-negative arthropathy; arthropathy; Wright's disease; psoriasis arthropathy; ulcerative colon arthropathy; enteric synovitis; chlamydia disease; Yersinia and Salmonella Associated arthropathy; Spinal arthropathy; Atherosclerosis / arteriosclerosis; Atopic allergy; Autoimmune bullous disease; Pemphigus vulgaris; Pemphigus decidans; Pemphigoid; Linear IgA disease; Autologous Immune hemolytic anemia; Qom's positive hemolytic anemia; acquired malignant anemia; juvenile malignant anemia; myalgia encephalitis / royal free disease; chronic mucosal skin candidiasis; macroscopic Cell arteritis (GCA); Primary sclerosing hepatitis; Cryptogenic autoimmune hepatitis; Acquired immune deficiency syndrome (AIDS); Acquired immune deficiency-related diseases; Hepatitis B; Hepatitis C; General variant immunodeficiency (General variant hypogammaglobulinemia); dilated cardiomyopathy; female infertility; ovarian failure; premature ovarian failure; fibrotic lung disease; cryptogenic fibrotic alveolitis; interstitial lung disease after inflammation; interstitial Pneumonia; Connective tissue disease-related interstitial lung disease; Mixed connective tissue disease-related lung disease; Systemic sclerosis-related interstitial lung disease; Rheumatoid arthritis-related interstitial lung disease; Systemic lupus erythematosus-related Pulmonary disease; Dermatomyositis / polymyositis-associated lung disease; Sjogren's disease-associated lung disease; Ankylosing spondylitis-associated lung disease; Vasculitis diffuse lung disease; Hemosiderin-associated lung disease; Drug-induced interstitial Fibrosis; Fibrosis; Radiofibrosis; Obstructive bronchiolitis; Chronic eosinophilic pneumonia; Lymphocytic invasive lung disease; Interstitial lung disease after infection; Gouty joints Inflammation; autoimmune hepatitis; type 1 autoimmune hepatitis (typical autoimmune hepatitis or lupus-like hepatitis); type 2 autoimmune hepatitis (anti-LKM antibody hepatitis); autoimmune-mediated hypoglycemia; accompanied by Type B insulin resistance of acanthosis nigricans; parathyroid hypokinesis; osteoarthritis; primary sclerosing cholangitis; type 1 cowhide Ringworm; type 2 psoriasis; idiopathic leukopenia; autoimmune neutropenia; NOS nephropathy; filamentous nephritis; renal microangiitis; Lyme disease; discoid lupus erythematosus; idiopathic Male infertility; nitric oxide-related male infertility; sperm autoimmune disease; multiple sclerosis (all subtypes, including primary progressive, secondary progressive, relapsing-remitting); sympathetic ophthalmia; Hypercirculatory blood pressure secondary to connective tissue disease; Good Paster syndrome; pulmonary manifestations of nodular polyarteritis; acute rheumatic fever; rheumatoid spondylitis; Still's disease; systemic sclerosis Huguelian's syndrome; High's disease / arteritis; autoimmune thrombocytopenia (AITP); idiopathic thrombocytopenia; autoimmune thyroid disease; hyperthyroidism; goiter autoimmune hypothyroidism Disease (Hashimoto's disease); atrophic autoimmune thyroid hypofunction; primary myxedema; lens-derived uveitis; primary vasculitis; white spot disease; acute liver disease; chronic liver disease; alcoholic cirrhosis Alcohol-induced liver damage; cholestasis; specific constitutional liver disease; drug-induced hepatitis; non-alcoholic steatohepatitis; allergies; group B streptococcus (GBS) infection; mental disorders (such as depression and schizophrenia) Th2 and Th1 mediated diseases; acute and chronic pain (different forms of pain); cancer (such as lung, breast, stomach, bladder, colon, pancreas, ovarian, prostate and rectal cancer) Hematopoietic malignant disease; Leukemia; Lymphoma; Beta lipoproteinemia; Hand and foot cyanosis; Acute and chronic parasitic or infectious processes; Acute leukemia; Acute lymphoblastic leukemia (ALL); T cell ALL; FAB ALL; Acute bone marrow Leukemia (AML); Acute or chronic bacterial infection; Acute pancreatitis; Acute renal failure; Adenocarcinoma; Atrial ectopic beat; AIDS dementia syndrome; Alcohol-induced hepatitis; Allergic conjunctivitis; Allergic contact dermatitis; Allergic Rhinitis; allograft rejection; alpha-1 antitrypsin deficiency; amyotrophic lateral sclerosis; anemia; angina pectoris; anterior horn cell degeneration; anti-CD3 therapy Antiphospholipid syndrome; antireceptor allergic reaction; aortic and peripheral aneurysms; aortic dissection; arterial hypertension; arteriosclerosis; arteriovenous fistula; ataxia; atrial fibrillation (persistent or paroxysmal); Atrial flutter; atrioventricular block; B-cell lymphoma; Bone graft rejection; Bone marrow transplantation (BMT) rejection; bundle branch block; Burkitt's lymphoma; burns; arrhythmia; cardiac stun syndrome; cardiac tumors; cardiomyopathy; heart and lung bypass inflammation; cartilage transplant rejection Cerebellar cortical degeneration; Cerebellar disorders; Disordered or multifocal atrial tachycardia; Chemotherapy-related disorders; Chronic myelogenous leukemia (CML); Chronic alcoholism; Chronic inflammatory lesions; Chronic lymphocytic leukemia ); Chronic obstructive pulmonary disease (COPD); Chronic salicylate poisoning; Colorectal cancer; Congestive heart failure; Conjunctivitis; Contact dermatitis; Pulmonary heart disease; Coronary artery disease; Cuzard's disease; Culture-negative sepsis; Cystic fibrosis; Cell hormone therapy-related conditions; Boxer dementia; Demyelination; Hemorrhagic dengue; Dermatitis; Skin conditions; Diabetes; Diabetic arteriosclerosis; Generalized Lewy body disease; Dilated congestive Cardiomyopathy; Basal ganglia disorders; Middle-aged Down's syndrome; Drug-induced by drugs blocking CNS dopamine receptors Dyskinesias; drug allergies; eczema; encephalomyelitis; endocarditis; endocrine diseases; epiglottis; Epstein-Barr virus infection; erythematous limb pain; extrapyramidal and cerebellar disorders; familial hemophagolymphoid tissue Hyperplasia; fetal thymic implant rejection; Friedreich's ataxia; functional peripheral arterial disease; fungal sepsis; gas gangrene; gastric ulcer; glomerulonephritis; transplant rejection of any organ or tissue Gram-negative sepsis; Gram-positive sepsis; granulomas caused by intracellular organisms; hairy cell leukemia; Harley-Davidson disease; Hashimoto's thyroiditis; hay fever; heart transplant rejection; hemochromatosis Hemodialysis hemolytic uremic syndrome / thrombotic thrombocytopenic purpura hemorrhage hepatitis A Heath bundle arrhythmia HIV infection / HIV neuropathy Hodgkin's disease hyperactivity dyskinesia allergic Reaction; allergic pneumonia; hypertension; hypokinesia; hypothalamic-pituitary-adrenal axis assessment; idiopathic Addison's disease; idiopathic lung Vitification (IPF); Antibody-mediated cytotoxicity; Weakness; Infant spinal muscular atrophy; Aortic inflammation; Influenza A; Exposure to ionizing radiation; Iris ciliaryitis / Uveitis / Optic neuritis; Ischemia -Reperfusion injury; ischemic stroke; juvenile rheumatoid arthritis; juvenile spinal muscular atrophy; Kaposi's sarcoma; Renal transplant rejection; Legionnaires' disease; Leishmaniasis; Leprosy; Cortical spinal cord system lesions; Fat edema; Liver transplant rejection; Lymphedema; Malaria; Malignant lymphoma; Malignant melanoma; Meningitis; Meningococcalemia; Metabolic syndrome; Migraine; Idiopathic migraine; Mitochondrial multisystem disorder; Mixed connective tissue disease; Single strain of gamma globulin disease; Multiple myeloma Multiple system degeneration (Manche, Dejein-Thomas, Shay-Drag, and Machado-Joseph); Myasthenia gravis; intracellular avian mycobacterium disease; Mycobacterium tuberculosis; bone marrow development Adverse syndromes; Myocardial infarction; Myocardial ischemic disorders; Nasopharyngeal carcinoma; Chronic neonatal lung disease; Nephritis; Nephropathy; Neurodegenerative diseases; Neurological type I muscle atrophy; Neutropenic fever; Non-Hodgkin's Lymphoma; occlusion of abdominal aorta and its branches; occlusive arterial disease; OKT3® therapy; bursitis / adjunctivitis; bursitis / vasanectomy; organ enlargement; osteoporosis Pancreatic transplant rejection; pancreatic cancer; tumor-associated signs / malignant hypercalcemia; parathyroid transplant rejection; pelvic inflammatory disease; perennial rhinitis; pericardial disease; peripheral atherosclerotic disease; peripheral vascular disease Peritonitis; malignant anemia; pneumocystis carinii pneumonia; pneumonia; POEMS syndrome (polyneuropathy, organ enlargement, endocrine disease, single gamma globulin disease and skin change syndrome); post-perfusion syndrome; post-pump syndrome; MI Symptoms after cardiac incision; preeclampsia; progressive supranuclear palsy; primary pulmonary hypertension; radiation therapy; Raynaud's phenomenon; Raynaud's disease; Ray Fussen's disease; regular narrow QRS tachycardia; high renal vascularity Blood pressure; Reperfusion injury; Restrictive cardiomyopathy; Sarcoma; Senile chorea; Lewy body Alzheimer's disease; Seronegative arthropathy; Shock; Sickle cell anemia; Skin allograft rejection; Skin change syndrome; Small bowel transplant rejection Response; solid tumor; special arrhythmia; spinal ataxia; spinal cerebellar degeneration; strep Myositis; cerebellar structural lesions; subacute sclerosing panencephalitis; syncope; cardiovascular system syphilis; systemic allergies; systemic inflammatory response syndrome; systemic onset juvenile rheumatoid arthritis; capillary dilatation; thromboocclusive Vasculitis; Thrombocytopenia; Toxicity; Transplantation; Trauma / bleeding; Type III allergic reaction; Type IV allergy; unstable colic; uremia; urinary septicemia; urticaria; heart valve disease; varicose veins; vasculitis; venous disease; venous thrombosis; ventricular fibrillation; virus and Fungal infections; viral encephalitis / aseptic meningitis; virus-associated hemophagocytic syndrome; Wernick-Korsakov syndrome; Wilson's disease; xenograft rejection of any organ or tissue; acute coronary syndrome; acute Idiopathic polyneuritis; acute inflammatory demyelinating polyradiculoneuropathy; acute ischemia; adult Still's disease; patchy alopecia; systemic allergic reaction; antiphospholipid antibody syndrome; aplastic anemia; arteries Sclerosis; atopic eczema; atopic dermatitis; autoimmune dermatitis; autoimmune disorders associated with streptococcal infection; autoimmune bowel disease; autoimmune hearing loss; autoimmune lymphoproliferative syndrome (ALPS) Autoimmune myocarditis; autoimmune premature ovarian failure; eyelid; bronchiectasis; bullous pemphigoid; cardiovascular disease; disaster Celiac disease; cervical spine joint adhesion; chronic ischemia; cicatricial pemphigoid; clinically isolated syndrome (CIS) with risk of multiple sclerosis; conjunctivitis; childhood-onset psychosis; lacrimal sac Dermatomyositis; diabetic retinopathy; disc herniation; disc herniation; drug-induced immune hemolytic anemia; endocarditis; endometriosis; endometritis; episcleritis; erythema polymorpha; Severe erythematous erythema; Pemphigoid gestation during pregnancy; Grignard-Barr's syndrome (GBS); Pollen fever; Hughes syndrome; Idiopathic Parkinson's disease; Idiopathic interstitial pneumonia; IgE-mediated allergies Disease; immune hemolytic anemia; inclusion body myositis; infectious ocular inflammatory disease; inflammatory demyelinating disease; inflammatory heart disease; inflammatory kidney disease; iritis; keratitis; dry keratoconjunctivitis; Or Kui-Mei's disease; Landry's palsy; Lange's cell histiocytosis; reticular leukoplakia; macular degeneration; microscopic polyangiitis; Behticelev's disease; motor neuron disorders; mucosa Pemphigoid; multiple organs Failure; myasthenia gravis; myelodysplastic syndrome; myocarditis; nerve root disorders; neuropathy; non-type A and non-B hepatitis; optic neuritis; osteolysis; oligoarticular JRA; peripheral arterial occlusive disease (PAOD); peripheral vascular disease (PVD); peripheral arterial disease (PAD); phlebitis; multiple nodular arteries Inflammation (or nodular periarteritis); polychondritis; rheumatic polymyalgia; gray hair; polyarticular JRA; polyendocrine syndrome; polymyositis; rheumatic polymyalgia (PMR); after pumping Syndromes; Primary Parkinson's disease; Secondary Parkinson's disease; Prostatitis; Pure red blood cell aplasia; Primary adrenal insufficiency; Recurrent optic neuromyelitis; Restenosis; Rheumatic heart disease; SAPHO (slip Meningitis, acne, pustular disease, osteoporosis and osteitis); secondary amyloidosis; shock lung; scleritis; sciatica; secondary adrenal insufficiency; polysiloxane related connective tissue disease; Weier's dermatosis; ankylosing spondylitis; Stephen-Johnson syndrome (SJS); systemic inflammatory response syndrome; temporal arteritis; toxoplasma retinitis; toxic epidermal necrolysis; transverse myelitis; TRAP (type 1 Tumor necrosis factor receptor (TNFR) -associated periodic syndrome); type B insulin resistance with acanthosis nigricans; type 1 allergic reaction; type 2 diabetes; urticaria; common interstitial pneumonia (UIP); Spring conjunctiva ; Retinitis; Vogt - Koyanagi - Harada syndrome (VKH syndrome); wet macular degeneration; wound healing; Jer coli and salmonella-related joint disease.

The binding protein of the present invention can be used to treat patients suffering from autoimmune diseases, especially those related to inflammation, rheumatoid arthritis (RA), osteoarthritis, psoriasis, multiple sclerosis (MS) and other autoimmune diseases. Disease of humans.

The antibody or antibody portion of the present invention may also be administered with one or more other therapeutic agents suitable for treating autoimmune and inflammatory diseases.

In one embodiment, diseases that can be treated or diagnosed with the compositions and methods of the present invention include, but are not limited to, primary and metastatic cancers, including breast cancer, colon cancer, rectal cancer, lung cancer, oropharyngeal cancer, hypopharynx Cancers of the department, esophagus, stomach, pancreas, liver, gallbladder and bile duct, small intestine, urinary tract (including kidney, bladder and urinary epithelial cancer), female reproductive tract cancer (including cervical cancer, uterus Cancer and ovarian cancer, choriocarcinoma and trophoblastic disease during pregnancy), male reproductive tract cancer (including prostate cancer, seminal vesicle cancer, testicular cancer and germ cell tumor), endocrine adenocarcinoma (including thyroid cancer, adrenal cancer and pituitary adenocarcinoma ) And skin cancer; And hemangiomas, melanomas, sarcomas (including sarcomas from bones and soft tissues and Kaposi's sarcoma), brain tumors, neurotumors, eye tumors, and meningeal tumors (including astrocytomas, gliomas, neuroglia Blastoma, retinoblastoma, neuroma, neuroblastoma, schwannomas, and meningiomas), solid tumors caused by hematopoietic malignancies such as leukemia, and lymphomas (Hodgkin's lymphoma and Non-Hodgkin's lymphoma).

In another embodiment, an antibody of the invention or an antigen-binding portion thereof is used to treat cancer or to prevent cancer metastasis from a tumor. The treatment may include administering the antibody or antigen-binding portion thereof, alone or in combination with another therapeutic agent or treatment, such as radiation therapy and / or chemotherapeutic agent.

The antibodies or antigen-binding portions thereof of the present invention may be combined with the following agents, including (but not limited to): anti-neoplastic agents, radiation therapy, chemotherapy, such as DNA alkylating agents, cisplatin, carboplatin ), Anti-tubulin agents, paclitaxel, docetaxel, paclitaxel, cranberries, gemcitabine, gemzar, anthracycline, adriamycin ), Topoisomerase I inhibitor, topoisomerase II inhibitor, 5-fluorouracil (5-FU), formazan tetrahydrofolate (leucovorin), irinotecan, receptor tyrosine kinase inhibition Agents (such as erlotinib, gefitinib), COX-2 inhibitors (such as celecoxib), kinase inhibitors, and siRNA.

The binding protein of the present invention can also be administered with one or more other therapeutic agents suitable for treating various diseases.

The antibodies or antigen-binding portions thereof of the present invention can be used alone or in combination to treat these diseases. It should be understood that the antibodies or antigen-binding portions thereof of the present invention may be used alone or in combination with other agents (such as therapeutic agents), which are selected by those skilled in the art for their intended purpose. For example, other agents may be therapeutic agents that are technically recognized as suitable for treating a disease or condition that can be treated by the antibodies of the invention. Other potions can also grant favorable attributes An agent for administering a therapeutic composition, such as an agent that affects the viscosity of the composition.

It should be further understood that the combinations intended to be included in the present invention are their combinations suitable for their intended purpose. The medicaments stated below are for illustrative purposes and are not intended to be limiting. The combination as part of the invention may be an antibody of the invention and at least one other agent selected from the list below. The combination may also include more than one other agent, such as two or three other agents, as long as the combination enables the formed composition to perform its intended function.

Preferred combinations are non-steroidal anti-inflammatory drugs (also known as NSAIDs), which include drugs such as ibuprofen. Other preferred combinations are corticosteroids, including prednisolone; the side effects that are well known for the use of steroids can be reduced or even eliminated by gradually reducing the steroid dose required when treating patients in combination with the anti-IL-1β antibodies of the invention. Non-limiting examples of therapeutic agents that can be used in combination with the antibodies or antibody portions of the invention for rheumatoid arthritis include, but are not limited to, the following: cytokine inhibitory anti-inflammatory drugs (CSAID); other human cytokines or growth factors Antibodies or antagonists, such human cytokines or growth factors are, for example, TNF, LT, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL- 8. IL-15, IL-16, IL-18, IL-21, interferon, EMAP-II, GM-CSF, FGF and PDGF. The antibodies or antigen-binding portions thereof of the present invention can be combined with antibodies directed against cell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1) , CD86 (B7.2), CD90, CTLA, or their ligands, including CD154 (gp39 or CD40L).

A preferred combination of therapeutic agents can interfere at different points in the autoimmune and subsequent inflammatory cascades; preferred examples include TNF antagonists such as chimeric, humanized or human TNF antibodies, D2E7 (PCT Publication No. WO 97/29131), CA2 (Remicade ^TM ), CDP 571 and soluble p55 or p75 TNF receptors, derivatives thereof p75TNFR1gG (Enbrel ^TM ) or p55TNFR1gG (lenacept), and TNFα converting enzyme (TACE) inhibitors; Similarly, other IL-1 inhibitors (interleukin-1 converting enzyme inhibitor IL-1RA, etc.) may be effective for the same reason. Other preferred combinations include interleukin-11. Another preferred combination is other key participants of the autoimmune response, which can function in parallel with, or function in conjunction with, IL-1β function. Another preferred combination is a non-consumable anti-CD4 inhibitor. Other preferred combinations include antagonists of the co-stimulatory pathway CD80 (B7.1) or CD86 (B7.2), including antibodies, soluble receptors or antagonist ligands.

The antibodies or antigen-binding portions thereof of the present invention can also be combined with the following agents, such as methotrexate, 6-MP, azathioprine, sulfasalazine, mesalazine, oxasalazine, chloroquinine / hydroxychloroquine (hydroxychloroquine), pencillamine, aurothiomalate (intramuscularly and orally), azathioprine, colchicine, corticosteroids (oral, inhaled and topical injection), β-2 adrenal Receptor agonists (salbutamol, terbutaline, salmeteral), xanthine (theophylline, aminophylline), cromoglycate ( cromoglycate, nedocromil, ketotifen, ipratropium and oxitropium, cyclosporine, FK506, rapamycin, mycophenolate morpholinate , Leflunomide, NSAID (e.g. ibuprofen), corticosteroids (e.g. prednisolone), phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents, complement inhibitors, adrenaline stimulants Interfering with signaling of pro-inflammatory cytokines such as TNF-α or IL-1 Agents (e.g. IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-1β converting enzyme inhibitors, TNFα converting enzyme (TACE) inhibitors, T cell signaling inhibitors (such as kinase inhibitors), metalloproteinase inhibition Agents, sulfasalazine, azathioprine, 6-mercaptopurine, angiotensin-converting enzyme inhibitors, soluble cytokine receptors and their derivatives (such as soluble p55 or p75 TNF receptors and derivatives p75TNFRIgG (Enbrel ^TM ) And p55TNFRIgG (Lanaxip), sIL-1RI, sIL-1RII, sIL-6R), anti-inflammatory cytokines (e.g. IL-4, IL-10, IL-11, IL-13 and TGFβ), celecoxib , Folic acid, hydroxychloroquine sulfate, rofecoxib, etanercept, infliximab, naproxen, valdecoxib, sulfasalazine, methylprednisolone, beauty Meloxicam, methylprednisolone acetate, gold sodium thiomalate, aspirin, triamcinolone acetonide, propoxyphene napsylate / apap, folic acid Salt, nabumetone, diclofenac, piroxicam, etodox Acid (etodolac), diclofenac sodium, oxaprozin, oxycodone hcl, hydrocodone bitartrate / apap, diclofenac sodium / misoprost Alcohol (misoprostol), fentanyl, human recombinant anakinra, tramadol hcl, salsalate, sulindac, cyanocobalamin (cyanocobalamin) / fa / pyridoxine, acetaminophen, alendronate sodium, prednisolone, morphine sulfate, lidocaine hydrochloride ), Indomethacin (glucosamine sulf / chondroitin), amitriptyline hydrochloride (amitriptyline hcl), sulfadiazine, oxycodone hydrochloride / acetaminophen Olopatadine hcl, misoprostol, naproxen sodium, omeprazole, cyclophosphamide, rituximab, IL-1 TRAP, MRA, CTLA4-IG, IL-18 BP, anti-IL-18, anti-IL15 BIRB-796, SCIO-469, VX-702, AMG-548, VX-740, roflumilast (Roflumilast), IC-485, CDC-801 and US Suopu Lan (Mesopram). Preferred combinations include methotrexate or leflunomide and, in the case of moderate or severe rheumatoid arthritis, cyclosporine.

Non-limiting other agents that can also be used in combination with binding proteins to treat rheumatoid arthritis (RA) include (but are not limited to) the following: non-steroidal anti-inflammatory drugs (NSAID); cytokine inhibitory anti-inflammatory drugs (CSAID); CDP- 571 / BAY-10-3356 (humanized anti-TNFα antibody; Celltech / Bayer); cA2 / infliximab (chimeric anti-TNFα antibody; Centocor); 75 kdTNFR-IgG / etanercept (75 kD TNF receptor -IgG fusion protein; Immunex; see, eg, Moreland et al. (Abstract No. 813), Arthritis Rheum., 37: S295 (1994); Baumgartner et al. , J. Invest. Med. , 44 (3): 235A (3 1996 Month)); 55 kdTNF-IgG (55 kD TNF receptor-IgG fusion protein; Hoffmann-LaRoche); IDEC-CE9.1 / SB 210396 (non-consumable primate anti-CD4 antibody; IDEC / SmithKline; see, for example, Kaine Et al. (Abstract No. 195), Arthritis Rheum., 38: S185 (1995)); DAB 486-IL-2 and / or DAB 389-IL-2 (IL-2 fusion protein; Seragen; see, for example, Sewell et al., Arthritis Rheum., 36 (9): 1223-1233 (September 1993)); anti-Tac (humanized anti-IL-2Rα; Protein Design Labs / Roche); IL-4 (anti-inflammatory cytokine; DNAX / Schering); IL-10 (SCH 520 00; recombinant IL-10, anti-inflammatory cytokines; DNAX / Schering); IL-4; IL-10 and / or IL-4 agonists (such as potent antibodies); IL-1RA (IL-1 receptor antagonist Synergen / Amgen); Anakin (Kineret ^® / Amgen); TNF-bp / s-TNF (soluble TNF-binding protein; see, for example, Evans et al. (Abstract No. 1540), Arthritis Rheum., 39 (9) (Supplement): S284 (1996); Kapadia et al., Amer . J. Physiol.-Heart and Circulatory Physiology, 268: H517-H525 (1995)); RP73401 (type IV phosphodiesterase inhibitor; see, for example, Chikanza et al. Human (Abstract No. 1527), Arthritis Rheum., 39 (9) (Supplement): S282 (1996)); MK-966 (COX-2 inhibitor; see, for example, Erich et al. (Abstract Nos. 328 and 329), Arthritis Rheum ., 39 (9) (Suppl): S81 (1996)); iloprost (iloprost on) (see, e.g., Scholz, P (Abstract No. 336.), Arthritis Rheum, 39 (9) ( Suppl):. S82 ( (1996)); methotrexate; thalidomide (see, for example, Lee et al. (Abstract No. 1524), Arthritis Rheum., 39 (9) (Suppl.): S282 (1996)) and thalidomide-related drugs (E.g. Celgen); Leflunomide (anti-inflammatory and cytokine inhibitors; see e.g. Fi nnegan et al. (Abstract No. 627), Arthritis Rheum., 39 (9) (Supplement): S131 (1996); Thoss et al., Inflamm. Res., 45: 103-107 (1996)); Tranexamic acid ( tranexamic acid) (plasminogen activation inhibitor; see, for example, Ronday et al. (Abstract No. 1541), Arthritis Rheum., 39 (9) (Suppl.): S284 (1996)); T-614 (cytokine inhibitor; See, for example, Hara et al. (Abstract No. 1526), Arthritis Rheum., 39 (9) (Supplement): S282 (1996)); Prostaglandin E1 (see, for example, Moriuchi et al. (Abstract No. 1528), Arthritis Rheum., 39 ( 9) (Supplement): S282 (1996)); Tenidap (non-steroidal anti-inflammatory drugs; see, for example, Guttadauria, M. (Abstract No. 1516), Arthritis Rheum., 39 (9) (Supplement): S280 (1996)); naproxen (non-steroidal anti-inflammatory drugs; see, for example, Fiebich et al., Neuro Report, 7: 1209-1213 (1996)); meloxicam (non-steroidal anti-inflammatory drug); ibuprofen (non-steroidal Anti-inflammatory drugs); piroxicam (non-steroidal anti-inflammatory drugs); diclofenac (non-steroidal anti-inflammatory drugs); indomethacin (non-steroidal anti-inflammatory drugs); sulfasalazine (see for example Farr et al. (Abstract No. 1519), Arthritis Rheum., 39 (9) (Supplement): S281 (1996)); Azathioprine (see, for example, Hickey et al. (Abstract No. 1521), Arthritis Rheum., 39 (9) (Supplement): S281 (1996)); ICE inhibitors ( Inhibitor of the enzyme interleukin-1β converting enzyme); zap-70 and / or lck inhibitors (inhibitors of tyrosine kinase zap-70 or lck); VEGF inhibitors and / or VEGF-R inhibitors (vascular Inhibitors of endothelial cell growth factor or vascular endothelial growth factor receptor; angiogenesis inhibitors); corticosteroid anti-inflammatory drugs (such as SB203580); TNF-converting enzyme inhibitors; anti-IL-12 antibodies; anti-IL-18 antibodies; Interleukin-11 (see e.g. Keith Jr. et al. (Abstract No. 1613), Arthritis Rheum., 39 (9) (Supplement): S296 (1996)); interleukin-13 (see e.g. Bessis et al. (Abstracts No. 1681), Arthritis Rheum., 39 (9) (supplement): S308 (1996)); interleukin-17 inhibitors (see, for example, Lotz et al. (Abstract No. 559), Arthritis Rheum., 39 (9) ( Supplement): S120 (1996)); gold; penicillamine; chloroquine; chlorambucil; hydroxychloroquine; cyclosporine; cyclophosphamide; total lymphatic irradiation; antithymocyte globulin; Anti-CD4 antibody; CD5-toxic ; Oral administration of peptides and collagen; lobenzarit disodium; cytokine regulators (CRA) HP228 and HP466 (Houghten Pharmaceuticals, Inc); ICAM-1 antisense phosphorothioate oligodesulfurization Oxynucleotide (ISIS 2302; Isis Pharmaceuticals, Inc); soluble complement receptor 1 (TP10; T Cell Sciences, Inc); prednisone; orgotein; glucosaminoglycan polysulfate Esters; minocycline; anti-IL2R antibodies; marine and plant lipids (fish and plant seed fatty acids; see, for example, DeLuca et al., Rheum. Dis. Clin. North Am. , 21: 759-777 (1995)) Auranofin; phenylbutazone; meclofenamic acid; flufenamic acid; intravenous immunoglobulin; zileuton; azalea Azaribine; mycophenolic acid (RS-61443); tacrolimus (FK-506); sirolimus (rapamycin); amiprilose (Xerofintin) (therafectin)); cladribine (2-chlorodeoxyadenosine); methotrexate; bcl-2 inhibitors (see Bruncko et al., J. Med. Chem., 50 (4), 641 -662 (2007)); antivirals and immunomodulators.

In one embodiment, a binding protein or an antigen-binding portion thereof is administered in combination with one of the following agents for treating rheumatoid arthritis (RA): a small molecule inhibitor of KDR, a small molecule inhibitor of Tie-2 Agents; methotrexate; prednisone; celecoxib; folic acid; hydroxychloroquine sulfate; rofecoxib; etanercept; infliximab; leflunomide; naproxen; vardecoxib Sulfasalazine; methylprednisolone; ibuprofen; meloxicam; methylprednisolone acetate; gold sodium thiomalate; aspirin; azathioprine; triamcinolone; propoxyphene naphthalenesulfonate / apap; folate; nabumetone; diclofenac; piroxicam; etodolac; diclofenac sodium; oxaprozine; oxycodone hydrochloride; dihydrocodeinone tartrate / apap; diclofenac / Misoprostol; fentanyl; human recombinant anakinin; tramadol hydrochloride; disalicylate; sulindac acid; cyanocobalamin / fa / pyridoxine; acetaminophen; alendronate Sodium; prednisolone; morphine sulfate; lidocaine hydrochloride; indomethacin; glucosamine sulfate / chondroitin; cyclosporine; Lin; Sulfadiazine; Oxycodone Hydrochloride / Acetaminophen Phenol; Olotadine Hydrochloride; Misoprostol; Naproxen Sodium; Omeprazole; Mycophenolate Morpholine Ethyl Ester; Cyclophosphamide; Toximab; IL-1 TRAP; MRA; CTLA4-IG; IL-18 BP; IL-12 / 23; anti-IL 18; anti-IL 15; BIRB-796; SCIO-469; VX-702; AMG-548 ; VX-740; Roflumilast; IC-485; CDC-801 and Methop Blue.

Non-limiting examples of therapeutic agents that can be used in combination with the binding proteins of the present invention for inflammatory bowel diseases include the following: Butinide; Epidermal Growth Factor; Corticosteroids; Cyclosporine; Salazine; Amine water Salicylate; 6-mercaptopurine; azathioprine; metronidazole; lipooxygenase inhibitor; mesalazine; oxasalazine; balsalazine; antioxidant; thromboxane inhibitor; IL-1 receptor Antagonists; anti-IL-1β mAb; anti-IL-6 mAb; growth factors; elastase inhibitors; pyridyl-imidazole compounds; antibodies or antagonists of other human cytokines or growth factors, such factors as TNF, LT , IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-17, IL-18, EMAP-II, GM-CSF, FGF and PDGF. The antibody or antigen-binding portion thereof of the present invention can be combined with an antibody of a cell surface molecule, such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90, or a ligand thereof. The antibodies or antigen-binding portions thereof of the present invention can also be combined with pharmaceuticals such as methotrexate, cyclosporine, FK506, rapamycin, mycophenolate morpholinate, leflunomide, NSAID ( (E.g. ibuprofen), corticosteroids (such as prednisolone), phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents, complement inhibitors, epinephrine activators, interference with pro-inflammatory cytokines ( Agents such as TNFα or IL-1) signaling (e.g. IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-1β converting enzyme inhibitors, TNFα converting enzyme inhibitors, T cell signaling inhibitors such as kinases Inhibitors), metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurine, angiotensin-converting enzyme inhibitors, soluble cytokine receptors and their derivatives (e.g., soluble p55 or p75 TNF receptors, sIL-1RI, sIL-1RII, sIL-6R) and anti-inflammatory cytokines (such as IL-4, IL-10, IL-11, IL-13 and TGFβ) and bcl-2 inhibitors.

Examples of therapeutic agents that can be combined with binding proteins for Crohn's disease include the following: TNF antagonists, such as the anti-TNF antibody adalimumab (PCT Publication No. WO 97/29131; HUMIRA®), CA2 (REMICADE) , CDP 571, TNFR-Ig construct, (p75TNFRIgG (ENBREL®) and p55TNFRIgG (LENERCEPT ^™ )) inhibitors and PDE4 inhibitors. The antibody or antigen-binding portion thereof of the present invention can be combined with a corticosteroid (such as butinide and dexamethasone). The binding protein or antigen-binding portion thereof of the present invention can also interact with drugs (such as sulfasalazine, 5-aminosalicylic acid, and oxasalazine) and interfere with the synthesis or action of pro-inflammatory cytokines (such as IL-1). Combination of pharmaceutical agents (such as IL-1 converting enzyme inhibitor and IL-1ra). The antibody or antigen-binding portion thereof of the present invention can also be used with a T cell signaling inhibitor (such as a tyrosine kinase inhibitor 6-mercaptopurine). The binding protein or antigen-binding portion thereof of the present invention can be combined with IL-11. The binding protein or the antigen-binding portion of the present invention can be used with mesalazine, prednisone, azathioprine, thiopurine, infliximab, methylprednisolone sodium succinate, diphenoxylate / atropine sulfate (atrop sulfate), loperamide hydrochloride, methotrexate, omeprazole, folate, ciprofloxacin / dextrose-water, codeine dihydrotartrate Ketones / apap, tetracycline hydrochloride, fluocinonide, metronidazole, thimerosal / boronic acid, cholestyramine / cholestyramine / sucrose, ciprofloxacin hydrochloride, scopolamine sulfate ( hyoscyamine sulfate, meperidine hydrochloride, midazolam hydrochloride, oxycodone hydrochloride / acetamide, promethazine hydrochloride, sodium phosphate, sulfamethoxamine Sulfamethoxazole / trimethoprim, celecoxib, polycarbophil, propoxyfen naphthalenesulfonate, hydrocortisone, multivitamin, balsalazine two (balsalazide disodium), codeine phosphate / apap, colesevelam hcl, cyanocobalamin, folic acid, levofloxacin, methylprednisolone, natalizumab And interferon gamma combination.

Non-limiting examples of therapeutic agents that can be used in combination with the binding protein of the present invention for multiple sclerosis (MS) include the following: corticosteroids; prednisolone; methylprednisolone; azathioprine Cyclophosphamide; cyclosporine; methotrexate; 4-aminopyridine; tizanidine; interferon-β1a (AVONEX; Biogen); interferon-β1b (BETASERON; Chiron / Berlex ); Interferon α-n3) (Interferon Sciences / Fujimoto), Interferon-α (Alfa Wassermann / J & J), Interferon β1A-IF (Serono / Inhale Therapeutics), PEGylated Interferon α2b (Enzon / Schering- Plough), copolymer 1 (Cop-1; COPAXONE; Teva Pharmaceutical Industries, Inc.); hyperbaric oxygen; intravenous immunoglobulin; cladribine; other human cytokines or growth factors and their receptors (such as TNF, LT , IL-1, IL-2, IL-6, IL-7, IL-8, IL-23, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF and PDGF) Antibodies or antagonists. The binding proteins of the present invention can be combined with antibodies to cell surface molecules such as CD2, CD3, CD4, CD8, CD19, CD20, CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, CD90 or their combinations Bit body. The binding proteins of the present invention can also be combined with agents such as methotrexate, cyclosporine, FK506, rapamycin, mycophenolate morpholinate, leflunomide, NSAID (e.g. ibuprofen ), Corticosteroids (such as prednisolone), phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents, complement inhibitors, adrenergic stimulants, interference with pro-inflammatory cytokines (such as TNFα or IL -1) Signaling agents (e.g. IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-1β converting enzyme inhibitors, TACE inhibitors, T cell signaling inhibitors (such as kinase inhibitors), metalloproteinases Inhibitors, sulfasalazine, azathioprine, 6-mercaptopurine, angiotensin-converting enzyme inhibitors, soluble cytokine receptors and their derivatives (e.g. soluble p55 or p75 TNF receptors, sIL-1RI, sIL- 1RII, sIL-6R), anti-inflammatory cytokines (such as IL-4, IL-10, IL-11, IL-13 and TGFβ) and bcl-2 inhibitors.

Examples of therapeutic agents that can be used in combination with the binding protein of the present invention for multiple sclerosis include interferon-β, such as IFNβ1a and IFNβ1b; copaxone; corticosteroids; 1 inhibitor; IL-1 inhibitor; TNF Inhibitors; and antibodies to CD40 ligands and CD80.

The binding proteins of the present invention can also be combined with pharmaceutical agents such as alenizumab, dronabinol, Unimed, daclizumab, mitoxantrone, xaliproden hydrochloride ), Fampridine, glatiramer acetate, natalizumab, sinnabidol, a-immune factor NNSO3, ABR-215062, AnergiX.MS, chemokine receptor Antagonist, BBR-2778, calagualine, CPI-1189, LEM (liposome-encapsulated mitoxantrone), THC.CBD (cannabinoid-like potentiator), MBP-8298, meso Puland (PDE4 inhibitor), MNA-715, anti-IL-6 receptor antibody, neurovax, pirfenidone allotrap 1258 (RDP-1258), sTNF-R1, talumpa Talampanel, teriflunomide, TGF-β2, tiplimotide, VLA-4 antagonists (e.g. TR-14035, VLA4-Ultrahaler, Antegran-ELAN / Biogen), interferon gamma Antagonist, IL-4 agonist.

Non-limiting examples of therapeutic agents that can be used in combination with the binding protein of the present invention for angina include the following: aspirin, nitroglycerin, isosorbide mononitrate, metoprolol succinate ), Atenolol, metoprolol tartrate, amlodipine besylate, diltiazem hydrochloride, isosorbide dinitrate, clopidogrel bisulfate (clopidogrel bisulfate), nifedipine, atorvastatin calcium, potassium chloride, furosemide, simvastatin, verapamil hcl, Digoxin, propranolol hydrochloride, carvedilol, lisinopril, spironolactone, hydrochlorothiazide, and maleic acid enala Enalapril maleate, nadolol, ramipril, enoxaparin sodium, heparin sodium, valsartan tan), salt Sotalol hydrochloride, fenofibrate, ezetimibe, bumetanide, losartan potassium, lisinopril / hydrochlorothiazide , Felodipine, captopril, bisoprolol fumarate.

Non-limiting examples of therapeutic agents that can be used in combination with the binding protein of the present invention for ankylosing spondylitis include the following: ibuprofen, diclofenac and misoprostol, naproxen, meloxicam, indomethacin, Diclofenac, celecoxib, rofecoxib, sulfasalazine, methotrexate, azathioprine, minocyclin, prednisone, etanercept, infliximab.

Non-limiting examples of therapeutic agents that can be used in combination with the binding proteins of the present invention for asthma include the following: abuterol, salmeterol / fluticasone, montelukast sodium , Fluticasone propionate, budesonide, prednisone, salmeterol hydroxynaphthoate, levalbuterol hcl, albuterol / isotropium sulfate, prednisone Sodium sodium phosphate, triamcinolone, beclomethasone dipropionate, ipratropium bromide, azithromycin, pirbuterol acetate, prednisolone, anhydrous Theophylline, methylprednisolone sodium succinate, clarithromycin, zafirlukast, formoterol fumarate, influenza virus vaccine, methylprednisolone, trihydrate Amoxicillin trihydrate, flunisolide, allergy injection, sodium cromoglycate, fexofenadine hydrochloride, mentho l), amoxicillin / clavulanate, levofloxacin, inhaler aids, guaifenesin, dexamethasone sodium phosphate, moxifloxacin hydrochloride, Doxycycline hyclate, guaifenesin / d-methorphan, p-ephedrine / cod / chlorphenir, gati Gatifloxacin, cetirizine hydrochloride, mometasone furoate, salmeterol hydroxynaphthoate, benzonatate, cephalexin, pe / Hydrocodone / clofenac, cetirizine hydrochloride / pseudoephed, phenylephrine / cod / promethazine, codeine / promethazine, cefprozil ), Dexamethasone, guaifenesin / pseudoephedrine, chlorpheniramine / hydrocodone, nedocromil sodium, terbutaline sulfate, epinephrine, methylprednisolone , Metaproterenol sulfate.

Non-limiting examples of therapeutic agents that can be used in combination with the binding proteins of the present invention for COPD include the following: abuterol sulfate / isotropium ammonium, ipratropium bromide, salmeterol / fluticasone, abuterol, hydroxynaphthalene Salmeterol formate, fluticasone propionate, prednisone, anhydrous theophylline, methylprednisolone sodium succinate, montelukast sodium, budesonide, formoterol fumarate, triamcinolone, levofloxacin , Guaifenesin, azithromycin, beclomethasone dipropionate, levobutrol hydrochloride, flunisolone, ceftriaxone sodium, amoxicillin trihydrate, gatifloxacin, zalustast , Amoxicillin / cobate salt, flunisolone / menthol, clofeniramine / hydrocodone, metaisoproterenol sulfate, methylprednisolone, mometasone furoate, p-ephedra / Cod / clofenac, piobuterol acetate, p-ephedrine / loratadine, terbutaline sulfate, tiotropium bromide, (R, R) -fu Moterol, TgAAT, Cilomilast, Roflumilast.

Non-limiting examples of therapeutic agents that can be used in combination with the binding proteins of the present invention for HCV include the following: interferon-α-2a, interferon-α-2b, interferon-α con1, interferon-α-n1, poly Pegylated interferon-α-2a, pegylated interferon-α-2b, ribavirin, pegylated interferon α-2b + ribavirin, ursodeoxycholic acid, licorice Glycyrrhizic Acid, Thymalfasin, Maxamine, VX-497 and subscripts through intervention Any compound that targets HCV: HCV polymerase, HCV protease, HCV helicase, HCV IRES (internal ribosome entry site).

Non-limiting examples of therapeutic agents that can be used in combination with the binding protein of the present invention for idiopathic pulmonary fibrosis include the following: prednisone, azathioprine, abutrol, colchicine, abutrol sulfate, digote Octane, interferon gamma, methylprednisolone sodium succinate, lorazepam, furosemide, lisinopril, nitroglycerin, spironolactone, cyclophosphamide, ipratropium bromide, Actinomycin d, alteplase, fluticasone propionate, levofloxacin, metaisoproterenol sulfate, morphine sulfate, oxycodone hydrochloride, potassium chloride, triamcinolone, tacrolimus anhydrous, calcium , Interferon-α, methotrexate, mycophenolate morpholinate, and interferon-γ-1β.

Non-limiting examples of therapeutic agents that can be used in combination with the binding protein of the present invention for myocardial infarction include the following: aspirin, nitroglycerin, metoprolol tartrate, enoxaparin sodium, heparin sodium, clopidogrel hydrogen sulfate Salt, carvedilol, atenolol, morphine sulfate, metoprolol succinate, warfarin sodium, lisinopril, isosorbide mononitrate, digoxin, furanilic acid , Simvastatin, ramipril, tenaiprise, enalapril maleate, torsemide, retavase, losartan potassium, quinapril hydrochloride / Mag carb, bumetanib, alteplase, enalaprilat, amiodarone hydrochloride, tirofiban hcl m-hydrate , Diltiazem hydrochloride, captopril, irbesartan, valsartan, propranolol hydrochloride, fosinopril sodium, lidocaine hydrochloride, eptifibatide , Cefazolin sodium, atropine sulfate, aminoca proic acid), spironolactone, interferon, sotalol hydrochloride, potassium chloride, docusate sodium, dobutamine hcl, alprazolam, pravastatin Sodium (pravastatin sodium), atorvastatin calcium, midazolam hydrochloride, pethidine hydrochloride, isosorbide dinitrate Esters, epinephrine, dopamine hydrochloride, bivalirudin, rosuvastatin, ezetimibe / simvastatin, avasimibe, cariporide ( cariporide).

Non-limiting examples of therapeutic agents that can be used in combination with the binding protein of the present invention for psoriasis include the following: small molecule inhibitors of KDR, small molecule inhibitors of Tie-2, calcipotriene, chlorofluoropropionate Clobetasol propionate, triamcinolone, halobetasol propionate, tazarotene, methotrexate, fluoxonol, enhanced betamethasone dipropionate, fluoride Fluocinolone acetonide, acitretin, tar shampoo, betamethasone valerate, mometasone furoate, ketoconazole, pramoxine / Fluocinolone, hydrocortisone valerate, flurandrenolide, urea, betamethasone, clobetasol propionate / emollient, fluticasone propionate Azithromycin, hydrocortisone, moisturizing formula, folic acid, desonide, pimecrolimus, coal tar, diflorasone diacetate, etanercept folic acid, lactic acid , Methoxsalen, hc / Bismuth gallate / znox / resor, methylprednisolone acetate, prednisone, sunscreen, halcinonide, salicylic acid, anthratriol, clocotolone pivalate ), Coal extract, coal tar / salicylic acid, coal tar / salicylic acid / sulfur, desoximetasone, diazepam, emollients, fluoxonol / emollients, mineral oil / Castor oil / na lactate, mineral oil / arachis oil, petroleum / isopropyl myristate, psoralen, salicylic acid, soap / tribromsalan, thimerosal / boronic acid, Celecoxib, Infliximab, Cyclosporine, Alfacept, efalizumab, Tacrolimus, Pimecrolimus, PUVA, UVB, Sulfasalazine.

Non-limiting examples of therapeutic agents that can be used in combination with the binding protein of the present invention for psoriasis arthritis include the following: methotrexate, etanercept, rofecoxib, celecoxib, leaves Acid, sulfasalazine, naproxen, leflunomide, methylprednisolone acetate, indomethacin, hydroxychloroquine sulfate, prednisone, sulindac acid, betamethasone dipropionate, infliximab Antibiotics, methotrexate, folate, triamcinolone, diclofenac, dimethylarsonine, piroxicam, diclofenac sodium, ketoprofen, meloxicam, methylprednisolone, nabumetone , Tolmetin sodium, calcipotriol, cyclosporine, diclofenac sodium / misoprostol, fluoxinol, glucosamine sulfate, gold sodium thiomalate, dihydrotartrate Indone / apap, ibuprofen, risedronate sodium, sulfadiazine, thioguanine, vardecoxib, afaset, efazumab, and bcl-2 inhibitors.

Non-limiting examples of therapeutic agents that may be used in combination with the binding proteins of the present invention for restenosis include the following: sirolimus, paclitaxel, everolimus, tacrolimus, zotalimus (Zotarolimus ), Acetamidophenol.

Non-limiting examples of therapeutic agents that can be used in combination with the binding protein of the present invention for sciatica include the following: dihydrocodeinone / apap, rofecoxib, cyclobenzaprine hcl, a Prednisone, naproxen, ibuprofen, oxycodone / acetamide, celecoxib, vardecoxib, methylprednisolone acetate, prednisone, codeine phosphate / apap, tramadol hydrochloride / Acetamide, methadone, meloxicam, methocarbamol, lidocaine hydrochloride, diclofenac, gabapentin, dexamethasone, carisoprodol, ketorolac Ketorolac tromethamine, indomethacin, acetaminophen, diazepam, nabumetone, oxycodone hydrochloride, tizanidine hydrochloride, diclofenac sodium / misoprostol, propoxyl naphthalenesulfonate Fen / apap, asa / oxycodone / oxycodone ter, ibuprofen / dihydrocodeinone tartrate, tramadol hydrochloride, etodolac, propoxyfen hydrochloride, amitriptyline hydrochloride , Myanthine / codeine phosphate / asa, morphine sulfate, multivitamins, naproxen sodium , Orphenadrine citrate, temazepam.

Examples of therapeutic agents that can be combined with the binding protein of the present invention for SLE (lupus) include the following: NSAID, such as diclofenac, naproxen, ibuprofen, piroxicam, indomethacin; COX2 inhibitors, such as Lenecoxib, rofecoxib, vardecoxib; antimalarials, such as hydroxychloroquine; steroids, such as prednisone, prednisone, butenad, dexamethasone; cytotoxins, such as azathioprine , Cyclophosphamide, mycophenolate morpholinate, methotrexate; PDE4 inhibitors or purine synthesis inhibitors, such as Cellcept. The binding proteins of the present invention can also interact with agents (such as sulfasalazine, 5-aminosalicylic acid, oxasalazine, azathioprine, Imuran) and interfere with pro-inflammatory cytokines (such as IL- 1) Combinations of synthetic, producing, or acting agents (eg, Cassin inhibitors, such as IL-1β converting enzyme inhibitors and IL-1ra). The binding protein of the present invention can also be used with T cell signaling inhibitors (such as tyrosine kinase inhibitors) or molecules that target T cell activating molecules (such as CTLA-4-IgG or anti-B7 family antibodies, anti-PD-1 family). Antibodies). The binding protein of the present invention can be used with IL-11 or anti-cytokine antibodies (such as fonotolizumab (anti-IFNg antibodies)) or anti-receptor receptor antibodies (such as anti-IL-6 receptor antibodies) and B cells Antibody combination of surface molecules. The antibody or antigen-binding portion thereof of the present invention can also be used with LJP 394 (abetimus), agents that deplete or inactivate B cells (such as rituximab (anti-CD20 antibody)), lymphostat-B ( (Anti-BlyS antibody), TNF antagonists (such as anti-TNF antibody adalimumab (PCT Publication No. WO 97/29131; HUMIRA®)), CA2 (REMICADE®), CDP 571, TNFR-Ig construct, (p75TNFRIgG (ENBREL®) and p55TNFRIgG (LENERCEPT®)) and bcl-2 inhibitors due to lupus-like phenotypes that have been shown to overexpress bcl-2 in transgenic mice (see Marquina et al., J Immunol. , 172 (11): 7177-7185 (2004)), so inhibition is expected to have a therapeutic effect.

A pharmaceutical composition of the invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antibody portion of the invention. A "therapeutically effective amount" means an amount effective to achieve a desired therapeutic result at the necessary dose and time period. The therapeutically effective amount of the antibody or antibody portion can be determined by those skilled in the art and can vary depending on factors such as the disease state, age, sex, and weight of the individual and the ability of the antibody or antibody portion to elicit a desired response in the individual. treatment An effective amount is also one in which a therapeutically beneficial effect exceeds any toxic or detrimental effect of the antibody or antibody portion. "Prophylactically effective amount" means an amount effective to achieve a desired preventive result at the necessary dose and time period. Generally, because a prophylactic dose is used in an individual before or early in the disease, a prophylactically effective amount is less than a therapeutically effective amount.

The dosing regimen can be adjusted to provide the best desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the urgency of the treatment situation. It is particularly desirable to formulate parenteral compositions into unit dosage forms to facilitate administration and achieve uniformity of dosage. A unit dosage form as used herein refers to a physical individual unit suitable as a single dose for a mammalian individual to be treated; each unit contains a predetermined amount of active compound and a required pharmaceutical carrier, which are calculated to produce the desired therapeutic effect. The specifications of the unit dosage form of the present invention are determined by and directly depend on the following factors: (a) the unique characteristics of the active compound and the specific therapeutic or preventive effect to be achieved, and (b) the active compound is mixed in the related technology for Inherent limitations of the sensitivity of the treated individual.

It should be noted that the dose value may vary depending on the type and severity of the condition to be alleviated. It should be further understood that for any particular individual, the particular dosing regimen should be adjusted over time based on individual needs and the professional judgment of the person who administers the composition or supervises the administration of the composition, and the dosage ranges set forth herein are merely exemplary It is not intended to limit the scope or operation of the claimed composition.

diagnosis

The disclosures in this article also provide diagnostic applications. This is further elucidated as follows. Antibodies that bind to the IL-1β of the present invention can be used in any of a variety of forms in vivo, in vitro, or ex vivo (i.e., in a cell or tissue obtained from a living individual, subject to a programmatic treatment, and Return to the subject) to detect IL-1β. The DVD-Ig of the present invention provides the further advantage of being able to bind to the epitopes of IL-1β and other antigens or epitopes in various diagnostic and detection assay formats.

I. Test method

The present invention also provides the use of at least one anti-IL-1β binding protein or antigen-binding portion thereof (including DVD-Ig) as described herein to determine the presence and amount of IL-1β or a fragment thereof ("analyte") in a sample. Or concentration method. Any suitable assay as known in the art can be used in the method. Examples include, but are not limited to, immunoassays, such as sandwich immunoassays (e.g., single strain, multiple strains, and / or DVD-Ig sandwich immunoassays, or any variation thereof (e.g., single strains / DVD-Ig, DVD-Ig / multiple strains) Etc.), including radioisotope detection (radioimmunoassay (RIA)) and enzyme detection (enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA) (e.g. Quantikine ELISA test, R & D Systems, Minneapolis, Minnesota)) ), Competitive inhibition immunoassay (such as forward and reverse), fluorescent polarization immunoassay (FPIA), enzyme doubling immunoassay technology (EMIT), bioluminescent resonance energy transfer (BRET), and homogeneous chemiluminescence assay. In a SELDI-based immunoassay, a capture reagent that specifically binds the relevant analyte (or a fragment thereof) is attached to the surface of a mass spectrometry probe, such as a pre-activated protein wafer array. The analyte (or a fragment thereof) is then specifically captured on a biochip, and the captured analyte (or a fragment thereof) is detected by mass spectrometric analysis. Alternatively, the analyte (or a fragment thereof) can be dissociated from the capture reagent and detected by conventional MALDI (matrix-assisted laser desorption / ionization) or by SELDI. Chemiluminescence microparticle immunoassay, especially an immunoassay using ARCHITECT® automatic analyzer (Abbott Laboratories, Abbott Park, Illinois) is an example of an exemplary immunoassay.

Methods for collecting, manipulating, and processing urine, blood, serum, plasma, and other body fluids well known in the art are used in the practice of the present invention, for example when using an anti-IL-1β binding protein as described herein for immunization Diagnostic reagents and / or when used in analyte immunoassay kits. The sample may contain other portions than the relevant analyte, such as antibodies, antigens, haptens, hormones, drugs, enzymes, receptors, proteins, peptides, polypeptides, oligonucleotides, and / or polynucleotides. For example, the sample may be a whole blood sample obtained from an individual. It may be necessary to treat a sample, especially whole blood, with a pretreatment reagent, for example, before an immunoassay as described herein. Necessary or needed. Even when pretreatment is not necessary (such as most urine samples), pretreatment can be performed as appropriate (such as part of a solution on a commercial platform).

The pretreatment reagent may be any reagent suitable for the immunoassay and kit of the present invention. Pre-treatment optionally includes: (a) one or more solvents (such as methanol and ethylene glycol) and optional salts, (b) one or more solvents and salts and optional detergents, (c) cleaning agents , Or (d) detergents and salts. Pretreatment reagents are known in the art and can be used, for example, in assays on Abbott TDx, AxSYM®, and ARCHITECT® analyzers (Abbott Laboratories, Abbott Park, Illinois), as in the literature (see, eg, Yatscoff et al., "Abbott TDx Monoclonal Antibody Assay Evaluated for Measuring Cyclosporine in Whole Blood", Clin.Chem. , 36: 1969-1973 (1990); and Wallemacq et al., "Evaluation of the New AxSYM Cyclosporine Assay: Comparison with TDx Monoclonal Whole Blood and EMIT Cyclosporine Assays ", Clin. Chem. , 45: 432-435 (1999)) and / or if commercially available, use this pretreatment. In addition, such as Abbott U.S. Patent No. 5,135,875, European Publication No. EP 0 471 293, PCT Publication No. WO 2008/082984, and U.S. Publication No. 2008/0020401 (the teachings on pretreatment are cited in their entirety) The methods described herein are incorporated herein). The pretreatment reagent may be a heterogeneous agent or a homogenizing agent.

Regarding the use of a heterogeneous pretreatment reagent, the pretreatment reagent precipitates an analyte-binding protein (such as a protein that can bind to an analyte or a fragment thereof) present in a sample. This pre-treatment step includes removing any analyte-binding protein by separating the supernatant of the mixture formed by adding the pre-treatment agent to the sample from the analyte-binding protein of Shendian. In this assay, any binding protein in the absence of the mixture supernatant is used in this assay, proceeding directly to the antibody capture step.

Regarding the use of homogeneous pretreatment reagents, this separation step does not exist. The entire mixture of sample and pretreatment reagent is contacted with a labeled specific binding partner of an analyte (or a fragment thereof), such as a labeled anti-analyte antibody (or an antigen-reactive fragment thereof). in Before or during capture by the first specific binding partner, the pretreatment reagents used in this assay are usually diluted in the pretreatment sample mixture. Despite this dilution, a certain amount of pretreatment reagent was still present (or retained) in the sample mixture during capture. According to the present invention, an exemplary labeled specific binding partner may be DVD-Ig (or a fragment, variant, or fragment of a variant thereof).

In a heterogeneous form, a first mixture is prepared after a sample is obtained from the individual. The mixture contains a sample for evaluating the analyte (or a fragment thereof) and a first specific binding partner, wherein the first specific binding partner and any analyte contained in the sample form the first specific binding partner Analyte-analyte complex. Preferably, the first specific binding partner is an anti-analyte antibody or a fragment thereof. The first specific binding partner may be DVD-Ig (or a fragment, variant, or fragment of a variant thereof) as described herein. The order in which the sample and the first specific binding partner are added to form the mixture is not critical. Preferably, the first specific binding partner is immobilized on a solid phase. The solid phase used in the immunoassay (for the first specific binding partner and optionally the second specific binding partner) may be any solid phase known in the art, such as (but not limited to) magnetic Particles, beads, test tubes, microtiter disks, colorimetric tubes, membranes, scaffold molecules, films, filter paper, discs and wafers.

After forming a mixture containing the first specific binding partner-analyte complex, any unbound analyte is removed from the complex using any technique known in the art. For example, unbound analytes can be removed by washing. However, ideally, the first specific binding partner is present in an amount exceeding any analyte present in the sample such that all analytes present in the sample are bound by the first specific binding partner.

After removing any unbound analytes, a second specific binding partner is added to the mixture to form a first specific binding partner-analyte-second specific binding partner complex. The second specific binding partner is preferably an anti-analyte antibody, which binds to an epitope on the analyte, which epitope is different from the first specificity on the analyte An epitope bound by a sexual binding partner. In addition, it is also preferred that the second specific binding partner is labeled with or contains the detectable label as described above. The second specific binding partner may be DVD-Ig (or a fragment, variant, or fragment of a variant thereof) as described herein.

Any suitable detectable label as known in the art may be used. For example, detectable labels can be radioactive labels (such as ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C, ³² P, and ³³ P), enzyme labels (such as horseradish peroxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase and its analogs), chemiluminescence labels (such as acridyl esters, thioesters, or sulfonamides; luminol, isoluminol, phenanthridinium esters, and the like Substances), fluorescent labels (such as luciferin (e.g. 5-luciferin, 6-carboxyfluorescein, 3'6-carboxyfluorescein, 5 (6) -carboxyfluorescein, 6-hexachloro- Luciferin, 6-tetrachlorofluorescein, fluorescein isothiocyanate and the like)), rhodamine, phycobiliprotein, R-phycoerythrin, quantum dots (e.g. zinc sulfide seals Covered with cadmium selenide), temperature-measuring markers, or immune polymerase chain reaction markers. An introduction to labeling, labeling procedures, and label detection can be found in Polak and Van Noorden, Introduction to Immunocytochemistry , 2nd Edition, Springer Verlag, NY (1997), and Haugland, Handbook of Fluorescent Probes and Research Chemicals (1996) (which is written by Molecular Probes, Inc., Eugene, Oregon combined manuals and catalogs). Fluorescent labels can be used in FPIA (see, for example, U.S. Patent Nos. 5,593,896, 5,573,904, 5,496,925, 5,359,093, and 5,352,803). Acridine compounds can be used as detectable labels in homogeneous or heterogeneous chemiluminescence assays (see, eg, Adamczyk et al., Bioorg. Med. Chem. Lett. , 16: 1324-1328 (2006); Adamczyk et al., Bioorg. Med. Chem. Lett. , 14: 2313-2317 (2004); Adamczyk et al., Biorg. Med. Chem. Lett. , 14: 3917-3921 (2004); and Adamczyk et al., Org. Lett. , 5: 3779-3782 (2003)).

An exemplary azridine compound is azrid-9-9 formamidine. The method for preparing acrid 9-formamidine is described in Mattingly, J. Biolumin. Chemilumin. , 6: 107-114 (1991); Adamczyk et al., J. Org. Chem. , 63: 5636-5639 (1998); Adamczyk et al., Tetrahedron , 55: 10899-10914 (1999); Adamczyk et al., Org. Lett. , 1: 779-781 (1999); Adamczyk et al., Bioconjugate Chem. , 11: 714-724 (2000); Adamczyk and Mattingly, Luminescence Biotechnology: Instruments and Applications ; (Dyke, KV) (CRC Press: Boca Raton, 2002) pp. 77-105; Adamczyk et al., Org. Lett. , 5: 3779-3782 (2003); And U.S. Patent Nos. 5,468,646, 5,543,524, and 5,783,699. Another preferred azine compound is azine-9-formate aryl ester. An example of an azine-9-formate aryl ester is 10-methyl-9- (phenoxycarbonyl) azine fluorosulfonate (commercially available from Cayman Chemical, Ann Arbor, Michigan). Methods for the preparation of acridine 9-formate aryl esters are described in McCarra et al., Photochem. Photobiol. , 4: 1111-21 (1965); Razavi et al., Luminescence , 15: 245-249 (2000); Razavi et al. Luminescence , 15: 239-244 (2000); and U.S. Patent No. 5,241,070. More details about acridine-9-formate aryl ester and its use are set forth in US Publication No. 2008/0248493.

Chemiluminescence assays (e.g., using acridine or other chemiluminescent agents as described above) can be performed according to the method described in Adamczyk et al., Anal . Chim . Acta , 579 (1): 61-67 (2006). Although any suitable assay format can be used, a microplate chemiluminometer (Mithras LB-940, Berthold Technologies USA, LLC, Oak Ridge, Tennessee) makes it possible to quickly verify multiple samples in small volumes.

The order in which samples and specific binding partners are added to form a chemiluminescence assay mixture is not critical. If the first specific binding partner is detectably labeled with a chemiluminescent agent, such as an acridine compound, a detectably labeled first specific binding partner-analyte complex is formed. Alternatively, if a second specific binding partner is used and the second specific binding partner is detectably labeled with a chemiluminescent agent (such as an acridine compound), a detectably labeled first specific binding partner is formed -Analyte-Second specific binding partner complex. Any unbound specific binding partner (whether labeled or unlabeled) It is removed from the mixture using any technique known in the art, such as washing.

Before, at the same time, or after the addition of the acridine compound described above, hydrogen peroxide may be generated in the mixture on site or hydrogen peroxide may be supplied or supplied to the mixture (e.g., the source of hydrogen peroxide is one or more known hydrogen peroxide-containing sources). Buffer or other solution). Hydrogen peroxide can be generated on-site in a variety of ways, such as those apparent to those skilled in the art.

Simultaneously or after adding at least one alkaline solution to the sample, a detectable signal, ie, a chemiluminescence signal, is generated indicating the presence of the analyte. The alkaline solution contains at least one base and has a pH value of 10 or more, preferably 12 or more. Examples of the alkaline solution include, but are not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonium hydroxide, magnesium hydroxide, sodium carbonate, sodium bicarbonate, calcium hydroxide, calcium carbonate, and calcium bicarbonate. The amount of alkaline solution added to the sample depends on the concentration of the alkaline solution. Based on the concentration of the alkaline solution used, those skilled in the art can easily determine the amount of alkaline solution added to the sample.

The generated chemiluminescence signal can be detected using conventional techniques known to those skilled in the art. Based on the intensity of the generated signal, the amount of analyte in the sample can be quantified. In particular, the amount of analyte in the sample is proportional to the intensity of the signal produced. The amount of analyte present can be quantified by comparing the amount of light produced to a standard curve of the analyte or to a reference standard. Standard curves can be generated by mass dilution analysis, gravimetric methods, and other techniques known in the art using serial dilutions of the analyte or solutions of known concentrations. Although the above description has focused on the use of an acridine compound as a chemiluminescent agent, a person of ordinary skill can easily adapt this description to use other chemiluminescent agents.

Analyte immunoassays can generally be performed using any format known in the art, such as, but not limited to, a sandwich format. In particular, in an immunoassay format, at least two antibodies are used to isolate and quantify analytes in a sample, such as human analytes or fragments thereof. More specifically, at least two antibodies bind to different epitopes on an analyte (or a fragment thereof) to form an immune complex, which is called a "sandwich". Generally, in immunoassays, one or more antibodies can be used to capture analytes (or fragments thereof) in a sample (these antibodies (Often referred to as "capture" antibodies) and one or more antibodies can be used to bind a detectable (i.e., quantitative) label to the sandwich (these antibodies are often referred to as "detection antibodies" or "conjugates"). Therefore, in the case of a sandwich immunoassay format, DVD-Ig (or a fragment, variant, or fragment thereof) as described herein can be used as a capture antibody, a detection antibody, or both. For example, a DVD-Ig with a first epitope domain on a bindable analyte (or a fragment thereof) can be used as a capture antibody and / or a second Another DVD-Ig of the epitope domain can be used as a detection antibody. In this regard, a DVD-Ig having a first domain that can bind a first epitope on an analyte (or a fragment thereof) and a second domain that can bind a second epitope on an analyte (or a fragment thereof) Can be used as a capture antibody and / or a detection antibody. Alternatively, a DVD-Ig having a first domain that can bind an epitope on a first analyte (or a fragment thereof) and a second domain that can bind an epitope on a second analyte (or a fragment thereof) can Used as capture antibodies and / or detection antibodies to detect and optionally quantify two or more analytes. If the analyte can be present in the sample in more than one form, such as monomeric and dimeric / multimeric (which can be homo- or hetero-meric), it has a One DVD-Ig of an epitope domain and another DVD-Ig having an epitope domain that can bind to a different part of a dimer / multimer form can be used as a capture antibody and / or a detection antibody, This makes it possible to detect and quantify different forms of a given analyte. In addition, the use of a DVD-Ig with differential affinities within a single DVD-Ig and / or between DVD-Ig can provide an affinity advantage. In the context of an immunoassay as described herein, it may generally be useful or desirable to incorporate one or more linkers into a DVD-Ig structure. When a linker is present, optimally, the linker should be of sufficient length and structural flexibility so that an epitope can be bound by the inner domain and another epitope can be bound by the outer domain. In this regard, if the DVD-Ig can bind two different analytes and one analyte is larger than the other, then the larger analyte is ideally bound by the outer domain.

In general, a sample that tests (e.g., suspected of containing) an IL-1β protein (or a fragment thereof) and at least one capture antibody and at least one detection antibody (which may be a second detection antibody or a first detection antibody) Three detection antibodies or even consecutively numbered antibodies, such as if the capture and / or detection antibodies comprise multiple antibodies) are contacted simultaneously or sequentially and in any order. For example, a sample may be contacted first with at least one capture antibody and then (sequentially) with at least one detection antibody. Alternatively, the sample may be contacted first with at least one detection antibody and then (sequentially) with at least one capture antibody. In another alternative, the sample may be contacted with both the capture antibody and the detection antibody at the same time.

In the sandwich assay format, a sample suspected of containing IL-1β (or a fragment thereof) is first contacted with at least one first capture binding protein (e.g., an IL-1β antibody) under conditions that allow the formation of a first binding protein / IL-1β complex. )contact. If more than one type of capture binding protein is used, a first capture binding protein / IL-1β complex including two or more types of capture binding proteins is formed. In the sandwich assay, the binding protein (ie, preferably at least one capture binding protein) is used in a molar excess of the maximum amount of the IL-1 β analyte (or fragment thereof) expected in the sample. For example, about 5 μg to about 1 mg of antibody per mL of buffer (eg, microparticle coating buffer) can be used.

Competitive inhibitory immunoassays, which are typically used to measure smaller analytes because of the need to bind only one antibody, include sequential and typical forms. In the sequential competitive inhibition immunoassay, the capture binding protein of IL-1β is coated on the well of a microtiter plate or other solid support. When a sample containing IL-1β is added to the well, IL-1β binds to the capture binding protein. After washing, a known amount of labeled (eg, biotin or horseradish peroxidase (HRP)) IL-1 β is added to the wells. Enzyme-labeled substrates are necessary to generate signals. An example of a suitable substrate for HRP is 3,3 ', 5,5'-tetramethylbenzidine (TMB). After washing, the signal produced by the labeled analyte is measured and is inversely proportional to the amount of IL-1β in the sample. In a typical competitive immunosuppression assay, a binding protein of IL-1β is coated on a solid support (such as a well of a microtiter plate). However, unlike sequential competitive suppression immunoassays, samples and labeled IL-1β are added to the wells simultaneously. Any IL-1β in the sample competes with the labeled IL-1β for binding to a capture binding protein. After washing, measure the amount of IL-1β The signal produced is inversely proportional to the amount of IL-1β in the sample.

Optionally, before contacting the sample with at least one capture binding protein (e.g., a first capture antibody), the at least one capture binding protein may bind to a solid support, which helps the first binding protein / IL-1β (or Fragment) complex is separated from the sample. The substrate to which the capture binding protein binds can be any suitable solid support or solid phase that facilitates separation of the capture antibody-analyte complex from the sample.

Examples include the wells of a plate (such as a microtiter plate), test tubes, porous gels (e.g., silicone, agarose, polydextrose, or gelatin), polymer films (e.g., polypropylene amidamine), beads (e.g., polystyrene beads) Or magnetic beads), strips of filter paper / membrane (e.g. nitrocellulose or nylon), particles (e.g. latex particles, magnetizable particles (e.g. with iron oxide or chromium oxide core and homo- or hetero-polymer coatings and 1-10 micron radius particles)). The substrate may comprise a suitable porous material with a suitable surface affinity for binding the antigen and a sufficient porosity to allow detection antibodies to reach. Although hydrated gel materials can be used, microporous materials are generally preferred. The porous substrates are preferably in the form of a sheet having a thickness of about 0.01 mm to about 0.5 mm, and preferably about 0.1 mm. Although the pore size can vary considerably, the pore size is preferably from about 0.025 microns to about 15 microns, and more preferably from about 0.15 microns to about 15 microns. These substrates can be activated by a chemical process that causes the antibody to covalently bond with the substrate. Generally, irreversible binding caused by the adsorption of an antigen or an antibody to a substrate through a hydrophobic force; or, chemical coupling agents or other methods can be used to covalently bind the antibody to the substrate, the limitation is that the binding will not interfere with the antibody binding Analyte capabilities. Alternatively, the antibodies can be bound to microparticles that have been previously treated with streptavidin (e.g. DYNAL® Magnetic Beads, Invitrogen, Carlsbad, California) or biotin (e.g. using Power-BindTM-SA-MP streptavidin Coated particles (Seradyn, Indianapolis, Indiana) or anti-species-specific monoclonal antibodies. If necessary, the substrate may be derivatized to allow reaction with various functional groups on the antibody. This derivatization requires the use of certain coupling agents, examples of which include, but are not limited to, maleic anhydride, N-hydroxysuccinimide, and 1-ethyl-3- (3-dimethylaminopropyl Carbonization II Imine. If desired, one or more capture reagents (such as antibodies (or fragments thereof)) each specific to the analyte can be attached to the solid phase at different physical or addressable locations (e.g., configured in a biochip (see, e.g., U.S. Patent No. No. 6,225,047; PCT Publication No. WO 99/51773; US Patent No. 6,329,209; PCT Publication No. WO 00/56934 and US Patent No. 5,242,828)). If the capture reagent is attached to a mass spectrometry probe as a solid support, the amount of analyte bound to the probe can be detected by laser desorption ionization mass spectrometry. Alternatively, a single column may be packed with different beads that are derived from the capture reagent or capture reagents, thereby capturing the analyte in a single location (see antibody-derived, bead-based technologies such as Luminex (Austin , Texas) xMAP technology).

After contacting a sample of the analyte (or fragment thereof) to be assayed with at least one capture antibody (e.g., a first capture antibody), the mixture is incubated to allow the formation of a first antibody (or multiple antibodies) -analyte (or a fragment thereof). )Complex. Incubation can be at a pH of about 4.5 to about 10.0, at a temperature of about 2 ° C to about 45 ° C, and for at least about 1 minute to about 18 hours, preferably about 1 minute to about 24 minutes, and most preferably about 4 minutes. Minutes to approximately 18 minutes. The immunoassay described herein can be performed in one step (meaning that the sample, at least one capture antibody, and at least one detection antibody are all added to the reaction container sequentially or simultaneously) or in more than one step (such as two steps, three steps Etc.).

After the (first or more) capture antibody / analyte (or fragment thereof) complex is formed, the complex is subsequently allowed to form (first or more) capture antibody / analyte (or fragment thereof) / second detection The test antibody complex is contacted with at least one detection antibody. Although described as a "secondary" antibody (for example, a second detection antibody) for clarity, in practice where multiple antibodies are used for capture and / or detection, at least one detection antibody may be used for The second, third, and fourth antibodies in the immunoassay. If the capture antibody / analyte (or fragment thereof) complex comes into contact with more than one detection antibody, a (first or more) capture antibody / analyte (or fragment thereof) / (multiple) detection antibody complex is formed. As with the capture antibody (e.g., the first capture antibody), when at least one (e.g., the second and any subsequent) detection antibody and capture antibody When the body / analyte (or fragment thereof) complex is contacted, a period of incubation under conditions similar to those described above is required to form (first or more) capture antibodies / analyte (or fragment thereof) / (second or more) ) Detection of antibody complexes. Preferably, at least one detection antibody contains a detectable label. A detectable label can be bound to at least one detection antibody (e.g., one, or more) of a capture antibody / analyte (or fragment thereof) / (second or more) detection antibody complex (e.g., Secondary detection antibody). Any detectable marker known in the art can be used (see discussion above, including Polak and Van Noorden (1997) and Haugland (1996) references).

The detectable label can be bound to the antibody directly or via a coupling agent. An example of a coupling agent that can be used is EDAC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride), which is commercially available from Sigma-Aldrich, St. Louis, Missouri . Other coupling agents that can be used are known in the art. Methods for binding detectable labels to antibodies are known in the art. In addition, many detectable labels, such as CPSP-azolidyl esters (i.e., 9- [N-tosylsulfonyl-N- (3-carboxypropyl)]-10- (3-sulfopropyl) acridine formamide) or SPSP-acridine ester (i.e.N10- (3-sulfopropyl) -N- (3-sulfopropyl) ) -Acridine-9-formamide).

The (first or more) capture antibody / analyte / (second or more) detection antibody complex can, but need not, be separated from the rest of the sample before quantitative labeling. For example, if at least one capture antibody (e.g., the first capture antibody) is bound to a solid support (such as a well or a bead), separation can be achieved by removing the (sample) fluid from contact with the solid support . Alternatively, if at least the first capture antibody is bound to a solid support, it can be simultaneously contacted with a sample containing the analyte and at least one second detection antibody to form a first (multiple) antibody / analyte / second (multiple) Antibody complex, and then remove the fluid (sample) to avoid contact with the solid support. If at least one first capture antibody does not bind to a solid support, the (first or more) capture antibody / analyte / (second or more) detection antibody complex need not be removed from the sample to quantify the labeled amount.

After forming a labeled capture antibody / analyte / detection antibody complex (e.g., a first capture antibody / analyte / second detection antibody complex), the complex is quantified using techniques known in the art The amount of the mark. For example, if an enzyme label is used, the labeled complex is reacted with the labeled substrate to produce a quantifiable reaction such as color development. If the label is radioactive, the label is quantified using an appropriate means, such as a scintillation counter. If the marker is a fluorescent marker, it stimulates the marker with one color of light (called the "excitation wavelength") and detects another color that the marker emits in response to the stimulus (called the "emission wavelength") To quantify the mark. If the label is a chemiluminescent label, the label is quantified by visual inspection or by detecting luminescence using a photometer, X-ray film, high-speed photographic film, CCD camera, or the like. Once the amount of labeling in the complex has been quantified, the concentration of the analyte or fragment thereof in the sample is determined by an appropriate method, such as by using a standard produced by serial dilutions of the analyte or fragment thereof of known concentration curve. In addition to using serial dilutions of the analyte or fragment thereof, standard curves can be generated gravimetrically, by mass spectrometry, and by other techniques known in the art.

In the chemiluminescence particle assay using an ARCHITECT® analyzer, the pH of the conjugate diluent should be approximately 6.0 +/- 0.2, and the particle coating buffer should be maintained at approximately room temperature (ie, approximately 17 ° C to approximately 27 ° C). The pH of the microparticle coating buffer should be about 6.5 +/- 0.2, and the pH of the microparticle diluent should be about 7.8 +/- 0.2. The solids are preferably less than about 0.2%, such as less than about 0.15%, less than about 0.14%, less than about 0.13%, less than about 0.12%, or less than about 0.11%, such as about 0.10%.

FPIA is based on the principle of competitive combined immunoassay. When a fluorescently labeled compound is excited by linearly polarized light, the fluorescently labeled compound will emit fluorescent light having a degree of polarization that is inversely proportional to its rotation rate. When the fluorescently labeled tracer-antibody complex is excited by linearly polarized light, the emitted light is still highly polarized because the rotation of the fluorophore between the time of absorbing light and the time of emitting light is limited. When a "free" tracer compound (i.e., a compound that does not bind to an antibody) is excited by linearly polarized light, its rotation ratio is less than that of competitive binding. Corresponding tracer-antibody conjugates produced during the immunization test are much faster. Because there is no radioactive material that requires special handling and disposal, FPIA is more advantageous than RIA. In addition, FPIA is a homogeneous test that can be performed easily and quickly.

In view of the above, a method for determining the presence, amount, or concentration of an analyte (or a fragment thereof) in a sample is provided. The method includes testing the analyte (or a fragment thereof) of the sample by: (i) using (i ') an antibody that can bind to the analyte, an antibody fragment, an antibody variant that can bind to the analyte, At least one of a fragment of an antibody variant that binds to the analyte and a DVD-Ig (or a fragment, variant or variant fragment thereof) that can bind to the analyte and (ii ') at least one detectable label, and (ii) a signal comprising a direct or indirect indication of the presence, amount, or concentration of an analyte (or a fragment thereof) in a sample generated by a detectable label and the analyte (or its Fragments) are compared for signals that are directly or indirectly indicative of the presence, amount or concentration. The calibrator is optionally part of a series of calibrators, where each calibrator is different from other calibrators based on the concentration of the analyte.

The method may include (i) contacting a sample with at least one first specific binding partner of an analyte (or a fragment thereof) to form a first specific binding partner / analyte (or fragment thereof) complex, the The at least one first specific binding partner is selected from the group consisting of an antibody that can bind to the analyte, an antibody fragment, an antibody variant that can bind to the analyte, an antibody variant fragment that can bind to the analyte, and a binding agent In the DVD-Ig (or fragment, variant, or variant fragment thereof) of the analyte, (ii) the first specific binding partner / analyte (or fragment thereof) complex with the analyte (or fragment thereof) At least one second specific binding partner is contacted to form a first specific binding partner / analyte (or a fragment thereof) / second specific binding partner complex, the at least one second specific binding partner is selected from the group consisting of A group consisting of a detectably labeled anti-analyte antibody that can bind to an analyte, a detectably labeled anti-analyte antibody fragment, and a detectably labeled anti-analyte antibody that can bind to an analyte Variant, may By bonding to the analyte of detectable labeled fragments of the variants of the anti-analyte antibody and the detectable label via the DVD-Ig (or a fragment, variant or variant sheet Paragraph), and (iii) by detecting or measuring the first specific binding partner / analyte (or fragment thereof) / second specific binding partner complex formed in (ii) The signal generated by the label can be detected to determine the presence, amount or concentration of the analyte in the sample. At least one first specific binding partner of the analyte (or a fragment thereof) and / or at least one second specific binding partner of the analyte (or a fragment thereof) is a DVD-Ig (or a fragment thereof) as described herein , Variant, or variant fragment) method may be preferred.

Alternatively, the method may include: contacting the sample with at least one first specific binding partner of the IL-1β analyte (or a fragment thereof), the at least one first specific binding partner being selected from the group consisting of: Antibodies, antibody fragments, antibody variants that can bind to the analyte, antibody variant fragments that can bind to the analyte, and DVD-Ig (or fragments, variants or variant fragments thereof) that can bind to the analyte; and simultaneously Or sequentially, in any order, the sample and at least one second specific binding partner that can compete with the analyte (or a fragment thereof) for binding to at least one first specific binding partner and selected from the group consisting of Contact: detectably labeled analyte that can bind to the first specific binding partner, detectable labeled analyte fragments, detectable labeled analysis that can bind to the first specific binding partner Variants, and fragments of detectably labeled analyte variants that can bind to the first specific binding partner. Any IL-1β (or fragment thereof) present in the sample competes with at least one second specific binding partner to form a first specific binding partner / analyte (or fragment thereof) complex and the first Specific binding partner / second specific binding partner complex. The method further comprises determining a signal in the sample by detecting or measuring a signal generated by a detectable label in the first specific binding partner / second specific binding partner complex formed in (ii). The presence, amount, or concentration of the analyte, where the signal generated by the detectable label in the first specific binding partner / second specific binding partner complex is inversely proportional to the amount or concentration of the analyte in the sample .

The method described above may further include diagnosing, predicting, or evaluating the efficacy of a therapeutic / prophylactic treatment of the patient from which the sample was obtained. If the method further includes assessing the For the efficacy of the therapeutic / prophylactic treatment, the method further includes optionally improving the therapeutic / prophylactic treatment of the patient to improve the efficacy as necessary. This method can be applied to automatic systems or semi-automatic systems.

As for the assay method (and its kit), a commercially available anti-analyte antibody or a method for generating an anti-analyte as described in the literature can be used. Suppliers of various antibodies include, but are not limited to, Santa Cruz Biotechnology Inc. (Santa Cruz, California), GenWay Biotech, Inc. (San Diego, California) and R & D Systems (RDS; Minneapolis, Minnesota).

In general, the predetermined content can be used as a benchmark by which to evaluate results obtained after assaying a sample of an analyte or a fragment thereof, for example, to detect a disease or disease risk. In general, in performing this comparison, by performing specific assays a sufficient number of times and under appropriate conditions such that a specific stage or endpoint of the presence, amount, or concentration of the analyte and the disease, disorder, or condition, or The association or association of signs to obtain a predetermined content. Generally, a predetermined content is obtained by testing a reference individual (or an individual population). The analytes measured may include fragments thereof, degradation products thereof, and / or enzymatic cleavage products thereof.

In particular, the amount or concentration of an analyte or a fragment thereof may be "unchanged", "favorable" (or "favorably changed") relative to a predetermined amount as used to monitor disease progress and / or treatment. , Or "adverse" (or "adversely changing"). "Elevated" or "increased" means that the amount or concentration in a sample is higher than a typical or normal content or range (such as a predetermined content) or higher than another reference content or range (such as a previous or baseline sample). The term "reduced" or "reduced" means that the amount or concentration in a sample is below a typical or normal content or range (such as a predetermined content) or below another reference content or range (such as a previous or baseline sample). The term `` change '' refers to a change (increase or decrease) in the amount or concentration in a sample compared to a typical or normal content or range (e.g., a predetermined amount) or to another reference content or range (e.g., a previous or baseline sample). ).

The typical or normal content or range of the analyte is defined according to standard practice. Because in In some cases, the content of the analyte is extremely low, so when there is any net change that cannot be explained by experimental error or sample deviation compared to the typical or normal content or range or reference content or range, it can be regarded as a so-called change has occurred Content or change. Therefore, the content measured in a particular sample will be compared to the content or range of content measured in a similar sample from a so-called normal individual. In this case, a "normal individual" is, for example, an individual who does not have a detectable disease, and a "normal" (sometimes referred to as a "control") patient or group is, for example, a patient or group that does not exhibit a detectable disease. In addition, assuming that the analytes are not routinely present in high levels in most human populations, a "normal individual" can be considered an individual that does not have a significant detectable increase or increase in amount or concentration of the analyte, and that "normal" (with ("Control" when used)) A patient or population is a patient or population that does not exhibit a significant detectable increase or increase in the amount or concentration of the analyte. "Apparently normal individuals" are individuals whose analytes have not been evaluated or are currently being evaluated. When the analyte is normally undetectable (eg, normal content is zero, or in the range of about 25 to about 75 percentiles of the normal population), but is detected in the sample, and when the analyte is When present in the sample at a higher than normal level, the analyte content is said to have "increased". Accordingly, the present invention provides, among other things, a method of screening individuals who are at or at risk for a particular disease, disorder, or condition. The test method may also involve the test of other markers and their analogs.

Therefore, the methods described herein can also be used to determine whether an individual has a specified disease, disorder, or condition or is at risk of developing a specified disease, disorder, or condition. In particular, the method may include the steps of: (a) determining the concentration or amount of IL-1β (or a fragment thereof) in a sample from an individual (e.g., using the methods described herein or known in the art Method); and (b) comparing the concentration or amount of IL-1β (or a fragment thereof) determined in step (a) with a predetermined content, wherein if the concentration or amount of the analyte measured in step (a) Advantageous relative to a predetermined amount, it is determined that the individual does not have the specified disease, disorder, or condition or is not at risk for the specified disease, disorder, or condition. However, if the concentration of IL-1β measured in step (a) If the degree or amount is unfavorable relative to the predetermined content, then it is determined that the individual has the specified disease, disorder or condition or is at risk of the specified disease, disorder or condition.

In addition, methods for monitoring the progress of disease in an individual are provided herein. Optimally, the method comprises the following steps: (a) determining the concentration or amount of IL-1β in a sample from the individual; (b) determining the concentration or amount of IL-1β in a later sample from the individual; And (c) comparing the concentration or amount of the analyte measured in step (b) with the concentration or amount of IL-1β measured in step (a), wherein if the concentration or amount measured in step (b) When the amount does not change or is unfavorable when compared with the concentration or amount of IL-1β determined in step (a), it is determined that the disease of the individual has continued, progressed, or worsened. In comparison, if the concentration or amount of IL-1β as determined in step (b) is advantageous when compared to the concentration or amount of IL-1β as determined in step (a), the individual's The disease has stopped, subsided or improved.

Optionally, the method further comprises comparing the concentration or amount of the IL-1β analyte as determined in step (b) with, for example, a predetermined content. Furthermore, if the comparison shows that the concentration or amount of the analyte as determined in step (b) is adversely changed, for example, relative to a predetermined content, the method optionally includes treating the individual with one or more pharmaceutical compositions for a period of time.

In addition, these methods can be used to monitor treatment in an individual treated with one or more pharmaceutical compositions. In particular, the methods include providing a first sample from the individual before the individual has administered one or more pharmaceutical compositions. Next, the concentration or amount of IL-Ι β in the first sample from the individual is determined (eg, using methods described herein or as known in the art). After the concentration or amount of IL-1β is determined, the concentration or amount of IL-1β is subsequently compared with a predetermined content as appropriate. If the concentration or amount of IL-1β as determined in the first sample is lower than the predetermined content, the individual is not treated with one or more pharmaceutical compositions. However, if the concentration or amount of IL-1β as determined in the first sample is higher than a predetermined content, the individual is treated with one or more pharmaceutical compositions for a period of time. The period of time for treating an individual with the pharmaceutical composition (s) may be determined by those skilled in the art (e.g., the period may be from about 7 days to about two years, preferably about 14 days to about 1 year).

During the course of treatment with the one or more pharmaceutical compositions, a second sample and subsequent samples are subsequently obtained from the individual. The number of samples and the time at which they are obtained from the entity are not critical. For example, a second sample may be obtained on day 7 after the first administration of the pharmaceutical composition to the individual, and a third sample may be obtained on the second week after the first administration of the pharmaceutical composition to the individual Obtained, the fourth sample can be obtained in the third week after the first administration of the pharmaceutical composition to the individual, the fifth sample can be obtained in the fourth week after the first administration of the pharmaceutical composition to the individual, etc. .

After each second or subsequent sample is obtained from the individual, the concentration or amount of the IL-1β analyte in the second or subsequent sample is determined (e.g., as described herein or as known in the art). method). The concentration or amount of IL-1β as determined in each of the second and subsequent samples will then be determined as in the first sample (e.g., a sample that is initially compared to a predetermined amount as the case may be). The concentrations or amounts of the analytes are compared. If the concentration or amount of IL-1β as determined in step (c) is advantageous when compared to the concentration or amount of the analyte as measured in step (a), then the individual's disease is judged to have ceased and subsided Or improvement, and the individual (s) of the pharmaceutical composition of step (b) should continue to be administered. However, if the concentration or amount determined in step (c) does not change or is unfavorable when compared to the concentration or amount of the analyte measured in step (a), it is determined that the individual's disease has continued, progressed, or Worsen, and the individual or the pharmaceutical composition administered to the individual in step (b) should be treated at a higher concentration or should be treated differently than one of the or the pharmaceutical composition administered to the individual in step (b) Or more pharmaceutical compositions to treat an individual. In particular, the individual may be treated differently from one or more of the pharmaceutical compositions or compositions previously received by the individual to treat or reduce the analyte content of the individual.

In general, for tests that are likely to be repeated (such as monitoring disease progression and / or treatment response), a second or subsequent sample is obtained at a time after the individual has obtained the first sample. In particular, a second sample from an individual may obtain a first sample from the individual Obtained in minutes, hours, days, weeks or years. For example, about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3 hours, About 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 Hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, About 6 days, about 7 days, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 Week, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, About 25 weeks, about 26 weeks, about 27 weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks, about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36 weeks, about 37 Week, about 38 weeks, about 39 weeks, about 40 weeks, about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45 weeks, About 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks, about 50 weeks, about 51 weeks, about 52 weeks, about 1.5 years, about 2 years, about 2.5 years, about 3.0 years, about 3.5 years, about 4.0 Years, about 4.5 years, about 5.0 years, about 5.5 years, about 6.0 years, about 6.5 years, about 7.0 years, about 7.5 years, about 8.0 years, about 8.5 years, about 9.0 years, about 9.5 years, or about 10.0 years A second sample was obtained from the individual at a time interval.

When used to monitor the course of disease, the assays described above can be used to monitor the course of disease in individuals suffering from acute conditions. Acute conditions, also known as critical care conditions, refer to acute life-threatening diseases or other critical medical conditions involving, for example, the cardiovascular system or the excretory system. In general, critical care conditions are those that require acute medical intervention in a hospital-based setting, including (but not limited to) an emergency room, intensive care unit, trauma center, or other emergency care setting, or through a nursing staff or other The symptoms of the care of medical staff. For critical care conditions, usually within a short period of time, i.e. minutes, hours or days (e.g. about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 60 Minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours , About 21 hours, about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days), and the initial test is the same Ground is usually carried out within a short period of time (e.g., about minutes, hours, or days) of the onset of a disease or condition.

Assays can also be used to monitor disease progression in individuals with chronic or non-acute conditions. Non-critical care or non-acute condition refers to a condition other than an acute, life-threatening disease or other critical medical condition involving, for example, the cardiovascular system and / or the excretory system. Generally, non-acute conditions include those of a longer or chronic duration. For non-acute conditions, usually over a longer period of time, such as hours, days, weeks, months or years (e.g. about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 Hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, About 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 2 About 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, About 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27 Week, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks, about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36 weeks, about 37 weeks, about 38 weeks, about 39 weeks, About 40 weeks, about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45 weeks, about 46 weeks, about 47 weeks, about 48 weeks About 49 weeks, about 50 weeks, about 51 weeks, about 52 weeks, about 1.5 years, about 2 years, about 2.5 years, about 3.0 years, about 3.5 years, about 4.0 years, about 4.5 years, about 5.0 years, about 5.5 Years, about 6.0 years, about 6.5 years, about 7.0 years, about 7.5 years, about 8.0 years, about 8.5 years, about 9.0 years, about 9.5 years, or about 10.0 years), and the initial test is also usually performed at A longer period of onset of the disease or condition (e.g. approximation Hours, days, months, or years).

In addition, the above assay may be performed using a first sample obtained from an individual, wherein the first sample is obtained from a source such as urine, serum, or plasma. Optionally, the above test may be subsequently repeated using a second sample obtained from the individual, wherein the second sample is obtained from another source. For example, if the first sample is obtained from urine, the second sample may be obtained from serum or plasma. The results obtained from the self-test using the first and second samples can be compared. Comparisons can be used to assess the state of an individual's disease or condition.

In addition, the invention also relates to methods for determining whether an individual susceptible to or suffering from a specified disease, disorder, or condition will benefit from treatment. In detail, the present invention relates to methods and products for companion diagnostics of analytes. Therefore, the method of "monitoring the disease treatment of an individual" as described herein further optimally can also cover the selection or identification of candidates for therapy.

Therefore, in a specific embodiment, the present invention also provides a method of determining whether an individual suffering from or at risk of a specified disease, disorder, or condition is a candidate for therapy. In general, an individual is someone who has experienced or is actually diagnosed with or at risk of a specified disease, disorder, or condition, and / or exhibits An individual having an adverse concentration or amount of an analyte or fragment thereof as described herein.

The method optionally includes an assay as described herein, before treating the individual with one or more pharmaceutical compositions (e.g., especially with a pharmaceutical agent related to the mechanism of action involving the analyte), immunosuppressive therapy, or immunosorbent therapy. IL-1β is evaluated and thereafter, or where the analyte is evaluated after the treatment and the concentration or amount of the analyte is compared with a predetermined content. An adverse concentration or amount of IL-1β observed after treatment confirms that the individual will not benefit from receiving further or continuous treatment, and a favorable concentration or amount of analyte observed after treatment confirms that the individual will benefit from receiving further or continuous treatment. This confirmation helps manage clinical studies and provides improved patient care.

It goes without saying that although certain embodiments herein are used to evaluate It is advantageous to identify diseases, disorders, or conditions, but assays and sets can be used to evaluate analytes in other diseases, conditions, and conditions. The test method may also involve the test of other markers and their analogs.

The assay method can also be used to identify compounds that improve a given disease, disorder, or condition. For example, a cell expressing an analyte can be contacted with a candidate compound. The expression level of the analyte in the cells in contact with the compound can be compared to the expression level in control cells using the assay methods described herein.

II. Sets

Kits for verifying the sample for the presence, amount, or concentration of the analyte (or a fragment thereof) in the sample are also provided. The kit contains at least one component of the IL-1β (or fragment thereof) for the test sample and instructions for the analyte (or fragment thereof) of the test sample. The component of at least one analyte (or fragment thereof) used to characterize the sample may include an anti-IL-1β binding protein (such as a monoclonal antibody or DVD-Ig ( Or a fragment, variant, or variant fragment thereof)).

The kit may include at least one component for testing an IL-1β analyte of a sample by an immunoassay (e.g., chemiluminescence microparticle immunoassay) and a test for immunoassay (e.g., chemiluminescence microparticle immunoassay). Instructions for the same IL-1β analyte. For example, the kit can include at least one specific binding partner of IL-1β, such as an anti-IL-1β single / multiple antibody (or a fragment thereof that can bind to an IL-1β analyte, which can bind to an assay A variant of the substance, or a variant fragment that can be bound to the analyte) or anti-IL-1β DVD-Ig (or a fragment, variant, or variant fragment thereof), either of which can be detectably labeled. Alternatively or in addition, the kit may comprise a detectably labeled IL-1β analyte (or it may bind to an anti-analyte monoclonal / multiple antibody or an anti-analyte DVD-Ig (or a fragment, variant or variant thereof) Fragment), which can compete with any analyte in the sample to bind to the single or multiple antibodies against the analyte (or a fragment that can bind to the analyte, a variant that can bind to the analyte, Or variant fragments that can bind to the analyte) or anti-analyte DVD-Ig (or fragments, variants or variants thereof) Allogeneic fragments), either of which can be fixed on a solid support. A kit can include a calibrator or control, such as an isolated or purified analyte. The kit may include at least one container (e.g., tube, microtiter tray or strip, which may have been coated with, for example, a first specific binding partner) for performing an assay and / or a buffer, such as an assay buffer or a wash Buffers, any of which can be provided as a concentrated solution, a substrate solution, or a stop solution of a detectable label (eg, an enzyme label). Preferably, the kit contains all components necessary for performing the assay, that is, reagents, standards, buffers, diluents, and the like. The instructions may be in paper form or computer readable form, such as a magnetic disk, CD, DVD, or the like.

Any binding protein (such as an anti-IL-1β binding protein or anti-analyte DVD-Ig or tracer) can be incorporated into a detectable label as described herein, such as a fluorophore, a radioactive moiety, an enzyme, a biotin / antibody Biotin protein labels, chromophores, chemiluminescent labels, or the like, or kits can include reagents for detectable labeling. Antibodies, calibrators, and / or controls can be provided in separate containers or pre-dispensed into appropriate assay formats, such as into microtiter plates.

Optionally, the set includes quality control components (such as sensitivity kits, calibrators, and positive controls). The preparation of quality control reagents is well known in the art and is described on the inserts of various immunodiagnostic products. The members of the sensitivity kit are used to establish the performance characteristics of the test as appropriate, and further, they are the applicable indicators of the completeness of the immunoassay kit and the standardization of the test.

The kit may also include other reagents, such as buffers, salts, enzymes, enzyme cofactors, enzyme substrates, detection reagents, and the like, as appropriate, for diagnostic tests or to assist in quality control assessments. Other components (such as buffers and solutions) that separate and / or process the sample (such as pretreatment reagents) can also be included in the kit. The kit may additionally include one or more other controls. One or more of the components of the kit may be lyophilized, in which case the kit may further comprise an agent suitable for reconstituting the lyophilized components.

Various components of the kit are provided in suitable containers as required, such as microdrops Fixed plate. The kit may further include a container (eg, a urine sample container or filter cartridge) for holding or storing a sample. Where appropriate, the kit may optionally contain a reaction vessel, a mixing vessel, and other components that facilitate the preparation of reagents or samples. The kit may also include one or more instruments that facilitate obtaining a sample, such as a syringe, pipette, forceps, measuring spoon, or the like.

If detectably labeled as at least one acrid compound, the kit may include at least one acrid-9-formamide, at least one acrid-9-formate aryl ester, or any combination thereof. If detectably labeled as at least one azidine compound, the kit may also include a source of hydrogen peroxide, such as a buffer, a solution, and / or at least one alkaline solution. If desired, the kit may contain a solid phase, such as magnetic particles, beads, test tubes, microtiter plates, cuvettes, membranes, scaffold molecules, films, filter paper, discs, or wafers.

III. Adaptability of packages and methods

Kits (or components thereof) and methods for determining the presence, amount, or concentration of an analyte in a sample by a test (such as an immunoassay) as described herein are applicable to, for example, U.S. Patent Nos. 5,089,424 and 5,006,309 A variety of automated and semi-automated systems (including systems in which the solid phase contains particles) are described and sold, for example, by Abbott Laboratories (Abbott Park, IL) as ARCHITECT®.

Some differences between automated or semi-automated systems compared to non-automated systems (e.g. ELISA) include the attachment of a first specific binding partner (e.g., an anti-analyte monoclonal / multiclonal antibody (or a fragment thereof, a variant thereof, or a variant thereof) Body fragments) or anti-analyte DVD-Ig (or fragments thereof, variants or variant fragments thereof)) substrates (in either way will affect sandwich formation and analyte reactivity), and capture, detect, and / Or the length and timing of any washing steps that may exist. Although non-automated formats (such as ELISA) may require a relatively long incubation time (e.g., about 2 hours) between sample and capture reagents, automatic or semi-automatic formats (e.g., ARCHITECT®, Abbott Laboratories) will have relatively short incubation times ( (For example, ARCHITECT®, about 18 minutes). Also, although non-automated formats (such as ELISA) may produce relatively long incubation times for detection antibodies (such as conjugate reagents) (E.g. about 2 hours), but an automatic or semi-automatic form (e.g., ARCHITECT®) will have a relatively short incubation time (e.g., about 4 minutes for ARCHITECT®).

Other platforms available from Abbott Laboratories include, but are not limited to, AxSYM®, IMx® (see, e.g., U.S. Patent No. 5,294,404), PRISM®, EIA (Beads) and Quantum ^™ II and other platforms. In addition, verification, kits, and kit components can be used in other forms, such as on electrochemical or other handheld or fixed-care verification systems. The present invention is applicable, for example, to a commercially available Abbott fixed-point care type (i-STAT®, Abbott Laboratories) electrochemical immunoassay system that performs a sandwich immunoassay. Immune sensors and methods of making them and operating them in disposable test devices are described in, for example, U.S. Patent No. 5,063,081, U.S. Pub. No. 0054078 and US Publication No. 2006/0160164.

In particular, regarding the adaptability of the analyte test to the I-STAT® system, the following configurations are preferred. Fabricate a micro-assembled silicon wafer with a pair of gold current analysis working electrodes and a silver-silver chloride reference electrode. On one working electrode, an anti-analyte single / multiple antibody (or a fragment thereof, a variant thereof or a variant fragment thereof) or an anti-analyte DVD-Ig (or a fragment thereof, a variant thereof or a variant thereof) will be immobilized Body fragments) of polystyrene beads (0.2 mm diameter) adhered to the polymer coating of patterned polyvinyl alcohol on the electrodes. This wafer is assembled into an I-STAT® cartridge in a fluidic form suitable for immunoassays. On a part of the chamber wall of the cartridge containing the sample, there are binding partners (such as anti-analyte single / multiple antibodies (or fragments thereof capable of binding to the analyte), variants thereof that are specific to the analyte Or a variant fragment thereof) or an anti-analyte DVD-Ig (or a fragment that can bind the analyte, a variant thereof, or a variant fragment thereof, which can all be detectably labeled). Inside the fluid bladder of the cartridge is an aqueous reagent including p-aminophenol phosphate.

In operation, a sample suspected of containing an analyte is added to the holding chamber of the test cartridge, and the cartridge is inserted into an I-STAT® reader. In combination with specific analytes After the formulation is dissolved in the sample, the pump element in the cartridge pushes the sample into the tube containing the wafer. Here, it is oscillated to promote the formation of a sandwich. In the penultimate step of the test, the fluid is pushed out of the fluid bladder and into a tube to wash the sample from the wafer and into the waste chamber. In the final step of the assay, the alkaline phosphatase tag reacts with p-aminophenol phosphate to cleave the phosphate group and allows the released p-aminophenol to be electrochemically oxidized at the working electrode. Based on the measured current, the reader is able to calculate the amount of analyte in the sample by means of an embedded algorithm and a calibration curve determined by the factory.

It goes without saying that the methods and kits described herein must encompass other reagents and methods for performing immunoassays. For example, various buffers are encompassed, such as known in the art and / or can be easily prepared or optimized for use, for example, in washing, as a conjugate diluent, particulate diluent, and / or as a calibrator Diluent buffer. Exemplary conjugate diluents are used in certain kits (Abbott Laboratories, Abbott Park, Illinois) and contain 2- (N-morpholinyl) ethanesulfonic acid (MES), salts, protein blockers, antimicrobials And cleaner ARCHITECT® conjugate thinner. Exemplary calibrator diluents are ARCHITECT® human calibrator diluents used in certain kits (Abbott Laboratories, Abbott Park, Illinois), which contain buffers containing MES, other salts, protein blockers, and antimicrobials . In addition, as described in US No. 12 / 650,241 (US Publication No. 2010/0167301; see also PCT Publication No. WO 2010/078443), for example, in the form of an I-Stat cartridge, a signal antibody can be used The nucleic acid sequence is used as a signal amplifier to obtain improved signal generation.

Those skilled in the art should readily understand that other suitable modifications and adaptations of the method of the invention described herein are obvious and can be performed using suitable equivalent forms without departing from the invention or the embodiments disclosed herein. category.

Now that the present invention has been described in detail, the present invention may be more clearly understood with reference to the following examples, which are included for illustrative purposes only and are not intended to limit the present invention.

Examples

Example 1: Production of affinity mature humanized IL-1β antibody from pure line E26

Table 6 provides the amino acid sequences of VH and VL of humanized mouse E26 antibody (GlaxoSmithKline, PCT Publication No. WO 95/01997). The amino acid residues of the individual CDRs of each VH and VL sequence are indicated in bold.

Affinity matured humanized mouse E26 antibody was obtained as follows. A light chain library was constructed to contain limited mutation induction at the following residues: CDRL1: 30, 31, 32; CDRL2: 50, 53, 55, 56; CDRL3: 92, 93, 94, 96, and 97 (Kabat numbering). Two heavy chain libraries were made to residues 31, 33, 50, 52a, 55, 56, 57, 58 and 60 (Kabat numbering) in CDRH1 and CDRH2 or residues 95, 96, 97 in CDRH3 , 98, 99, 100, 100a, 100b and 102 contained limited mutation induction. These heavy chain libraries are also at residues 23 (A / S), 24 (A / S), 62 (T / S), 84 (P / A), 88 (G / A), 91 (F / Y) Binary diversity at 108 and 108 (P / L) allows framework germline to be performed during library selection. All three libraries were independently selected by reducing the concentration of cyno IL-1β. All mutated CDR sequences were then combined in one library with mutations in only the VH CDRs and another library with mutations in all six CDRs. For human and cynomolgus IL-1β, the two combinatorial libraries were subjected to more stringent selection conditions, and individual antibodies were subsequently identified and expressed as IgG proteins for further characterization.

Table 7 provides a list of amino acid sequences derived from the VH region of the humanized E26 affinity matured IL-1 β antibody. The amino acid residues of the individual CDRs of each VH sequence are indicated in bold.

Table 8 provides a list of amino acid sequences derived from the VL region of the affinity mature humanized IL-1 β antibody of E26. The amino acid residues of the individual CDRs of each VL sequence are indicated in bold. The N-terminal D (Asp) to G (Gly) mutations seen in some of the affinity matured VL sequences in Table 8 below are most likely the result of unexpected mutation induction, which is induced by polymerase during library construction Occurs during a strand reaction (PCR). When using these regions to construct an IgG molecule, the N-terminal G residues can be removed recklessly.

The sequences of the individual CDRs from the VH and VL regions of the affinity matured IL-1 β antibodies of the humanized E26 in the table above can be aligned to provide common CDR sequences, such as the common CDR sequences in Table 9.

The sequences in Table 10 were converted into IgG for further characterization. Pure E26.13 mutations in the J region of the variable heavy and light chain regions (referred to as E26.13 JM VH and E26.13 JM VL) to remove non-germline framework mutations. The amino acid residues of individual CDRs are indicated in bold.

Example 2: Functional Characterization of IL-1β Antibodies

Example 2.1: IL-1β enzyme-linked immunosorbent assay scheme

To determine whether the anti-IL-1β mAb bound to human IL-1β, an ELISA plate (Nunc, MaxiSorp, Rochester, New York) and 2 μg / ml of an anti-human Fc antibody (Jackson Immunoresearch, West Grove) diluted with Pierce Coat buffer were used. , Pennsylvania) were incubated overnight at 4 ° C. The disks were washed 5 times with washing buffer (PBS containing 0.05% Tween 20) and blocked with 200 μl of Superblock blocking buffer (Thermo scientific, No. 37515) per well at 25 ° C. for 1 hour. The blocking buffer was removed by tapping the disk, and 2 μg / ml of each antibody in PBS containing 10% Superblock, 0.5% Tween-20 was added to each well at 100 μl per well at 25 ° C. Incubate for 1 hour. Each well was washed 5 times with 1 × PBST, and 1 μg / ml biotinylated antigen was titrated at a serial dilution of 1: 6 (for a range of μg to pg with PBS containing 10% Superblock, 0.05% Tween 20). Each dilution of the antigen was then added to the dish and incubated for 1 hour at 25 ° C. Each well was washed 5 times with 1 × PBST and incubated with polyHRP streptavidin (KPL No. 474-3000, Gaithersburg, Maryland) for 1 hour at 25 ° C. Each well was washed 5 times with 1 × PBST, and 100 μl of ULTRA-TMB ELISA (Pierce, Rockford, Illinois) was added to each well. After color development, the reaction was stopped with 1N HCL and the absorbance at 450 nm was measured. The results are shown in Table 11, and the values indicate that the anti-IL-1β antibody binds to human IL-1β.

Example 2.2: IL-1β antibody neutralizing potency

In order to study the functional activity of the anti-human IL-1β antibody in the present invention, the antibody was used in an MRC-5 assay that can measure the ability of the antibody to inhibit IL-1β activity. The MRC-5 cell line is a human lung fibroblast cell line that produces IL-8 in a dose-dependent manner in response to human IL-1β. This cell line also responds to cynomolgus IL-1β to produce IL-8. MRC-5 cells were originally obtained from ATCC and were subcultured in complete MEM at 10% FBS and grown in a 5% CO ₂ incubator at 37 ° C. To determine the neutralizing potency of the antibody against IL-1β, the antibody (50 μl) was added to a 96-well plate (final concentration range 1E-7 to 1E-15M) and 50 μl of human or human at 37 ° C, 5% CO ₂ Rhesus monkey IL-1 β (final concentration of 50 pg / mL) was pre-incubated together for 1 hour. The antigen-antibody complex (100 μL) was then added to MRC-5 cells (coated at 100 μl per well at a concentration of 1E5 / ml 24 hours ago). The assay plate was incubated overnight in a 5% CO ₂ incubator at 37 ° C. Antibody potency was measured by its ability to inhibit IL-8 production. Human IL-8 production was measured by a chemiluminescence-based assay. Table 12 summarizes the antibody efficacy on human and cynomolgus IL-1β.

ND: Not detected.

Example 2.3: Affinity measurement of IL-1β antibodies by surface plasmon resonance

The BIACORE test (Biacore, Inc, Piscataway, New Jersey) uses kinetics to measure association rate constants and dissociation rate constants to determine antibody affinity. Running HBS-EP (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20) at 25 ° C by using a Biacore® 3000 instrument (Biacore® AB, Uppsala, Sweden). Measurements based on surface plasmon resonance were performed to determine the binding of antibodies to purified recombinant human IL-1β and cynomolgus IL-1β. All chemicals were obtained from Biacore® AB (Uppsala, Sweden) or otherwise from different sources as described herein. According to the manufacturer's instructions and procedures, approximately 5000 RU goat anti-mouse IgG, (Fcγ), fragment-specific polyclonal antibodies (Pierce Biotechnology Inc, Rockford, Illinois) was directly fixed on a CM5 research-grade biosensor wafer at 25 μg / ml. Use ethanolamine to block unreacted parts on the surface of the biosensor. The modified carboxymethyl polyglucose surface in flow cells 2 and 4 was used as the reaction surface. Unmodified carboxymethyl polyglucose without goat anti-mouse IgG in flow cells 1 and 3 was used as a reference surface. In kinetic analysis, with the aid of Biaevaluation 4.0.1 software, the rate equations obtained from the 1: 1 Langmuir binding model were fitted to the association phases and dissociations of all eight injections (using the Overall fit analysis). The purified antibody was diluted with HEPES buffered saline to capture on the goat anti-human IgG specific reaction surface. The antibody (25 μg / ml) to be captured as a ligand was injected onto the reaction matrix at a flow rate of 5 μL / min. The association rate constant k _on (unit M ^-1 s ^-1 ) and the dissociation rate constant k _off (unit s ^-1 ) were measured at a continuous flow rate of 25 μL / min. Rate constants were obtained by measuring kinetic binding at ten different antigen concentrations in the range of 10-200 nM. Then, the equilibrium dissociation constant (unit M) of the reaction between the antibody and the target antigen is calculated according to the following automatic mechanical rate constant: K _D = k _off / k _on . The binding is recorded over time and the kinetic rate constant is calculated. In this test, the association rate as fast as 10 ⁶ M ^-1 s ^{-1 and} the dissociation rate as slow as 10 ^-6 s ^{-1 can} be measured. Table 13 shows the results of the affinity measurement of human anti-IL-1β antibodies.

ND: Not detected.

Example 3: IL-1α / β DVD-Ig ^TM Birth of molecules

Example 3.1: Construction of IL-1α / β DVD-Ig DNA construct

An anti-IL-1α antibody ("X3"; see PCT Publication No. WO 95/14780) variable domain and multiple IL-1β antibody variable domains are combined into a DVD by overlapping PCR amplification with an insert linker DNA sequence -Ig form (Wu et al ., Nature Biotechnol. , 25: 1290-1297 (2007); PCT Publication No. WO 2007/024715 A2). X3 is also mutated in the J region of the variable heavy and light chain regions (referred to as X3 JM VH and X3 JM VL, respectively) to remove non-germline framework mutations. The amplified PCR product was sub-selected into a performance vector suitable for transient expression in HEK293 cells and the open reading frame region was determined by sequencing before DVD-Ig expression.

Example 3.2: Expression and production of IL-1α / β DVD-Ig binding protein

After determining the DNA sequence, all DVD-Ig DNA constructs were amplified in E. coli and the DNA was purified using Qiagen Hispeed Maxi Prep (catalog number 12662, QIAGEN). DVD-Ig DNA was transfected to log-phase 293E cells (0.5 × 10 ⁶ / ml, by mixing PEI and DNA at a ratio of 2: 1 under 0.2 μg / ml heavy-chain DNA and 0.3 μg / ml light-chain DNA. (Viability> 95%). DNA: PEI complexes were formed in a TC cabinet at room temperature for 15 minutes and then added to 293E cells. After 20 hours, 0.5% TN1 to 293E cells were added. On day 5, the supernatant was collected for human IgG1 titer measurement. Cell supernatant was collected on day 7 and filtered through a 0.2 μM PES filter. The supernatant was purified by using Protein A Sepharose Affinity Chromatography according to the manufacturer's instructions. Purified DVD-Ig was eluted from the column with 0.1 M glycine (pH 2.99) and immediately dialyzed into 15 mM histidine buffer (pH 6.0). Binding proteins were quantified by A280 and analyzed by mass spectrometry and SEC.

Example 3.3: Sequence of IL-1α / β DVD-Ig Construct

The amino acid sequences of the heavy and light chains of DVD-Ig proteins capable of binding to human IL-1β and human IL-1α were determined. The amino acid sequences of the variable heavy chain (VH), variable light chain (VL), constant light chain (CL), and constant heavy chain (CH) regions of the IL-1α / β DVD-Ig binding protein are shown in the table below. 14 in. In Table 14, the amino acid sequences of the E26.13 and E26.35 VL regions are designated as SEQ ID NO: 238 and SEQ ID NO: 239, respectively, in place of SEQ ID NO: 205 and SEQ ID NO: 209 to explain the inclusion of the C-terminal arginine (R) residue. Those skilled in antibody engineering understand this C-terminal arginine residue as the amino acid residue at the junction of the VL and CL kappa regions of the IgG molecule, and it is sometimes included in the CL region or as shown in Table 14 below. In the VL region.

Linker sequences are indicated by underlined residues.

Example 4: Functional Characterization of IL-1α / β DVD-Ig Protein

Example 4.1: IL-1α / β enzyme-linked immunosorbent assay scheme

Evaluation of IL-1β / α DVD-Ig binding by ELISA (assay described above, Example 2.1) Protein binding to IL-1β and IL-1α. The results are shown in Table 15.

Example 4.2: IL-1α / β bioassay and neutralization test

MRC5 cells were coated in a volume of 100 μL at a density of 1.5-2 × 10 ⁴ cells per well and incubated overnight at 37 ° C. and 5% CO ₂ . 20 μg / mL DVD-Ig working stock solution (4-fold concentrated) was prepared in complete MEM medium. Eight-point serial dilutions (5 μg / mL-0.0003 μg / mL) were performed with complete MEM in a Marsh dilution plate. Add 65 μL of each antibody dilution and 65 μL of 200 pg / mL IL-1α or IL-1β solution or 65 μL of 50 pg / mL IL- to a 96-well v-bottom (Costar No. 3894) dish in quadruplicate. A mixed solution of both 1α and IL-1β solutions. Control wells received 65 μL of 200 pg / ml of IL-1α or IL-1β or 50 pg / mL of mixed IL-1α / β (4-fold concentrated) plus 65 μL of MEM medium and the medium control wells received 130 μL of medium. After 1 hour of incubation, 100 μL of DVD-Ig / Ag mixture was added to MRC5 cells. All pore volumes are equal to 200 μL. All disk reagents were then concentrated 1x. After 16-20 hours of incubation, the well contents (150 μL) were transferred to a 96-well round bottom plate (Costar No. 3799) and placed in a -20 ° C freezer. The hIL-8 content in the supernatant was tested by using a human IL-8 ELISA kit (R & D Systems, Minneapolis, Minnesota) or MSD hIL-8 (chemiluminescence kit). Neutralizing potency was calculated by calculating the percentage inhibition with respect to the IL-1α, IL-1β, or IL-1α / β control values alone (Table 16).

ND: Not detected.

Example 4.3: Affinity measurement of IL-1α / β DVD-Ig molecules

Binding of IL-1α / β DVD-Ig to purified recombinant human IL-1β and IL-1α and cynomolgus IL-1β and IL-1α was determined using surface plasmon resonance as described in Example 2.3 and the results are shown in In Table 17.

Incorporated by reference

The present invention incorporates, by reference in its entirety, techniques well known in the fields of molecular biology and drug delivery. Such techniques include, but are not limited to, those described in the following publications: Ausubel et al. (Eds.), Current Protocols in Molecular Biology , John Wiley & Sons, NY (1993); Ausubel, FM et al., Short Protocols In Molecular Biology (4th edition, 1999) John Wiley & Sons, NY. (ISBN 0-471-32938-X); Controlled Drug Bioavailability Drug Product Design and Performance , Smolen and Ball (eds.), Wiley, New York (1984) ); Giegé et al., Chapter 1, Crystallization of Nucleic Acids and Proteins, Practical Approach, 2nd Edition , (ed. Ducruix and Giegé) (Oxford University Press, New York, 1999) pp. 1-16; Goodson, JM, Chapter 6, Medical Applications of Controlled Release, Volume II, Applications and Evaluation , (Langer and Wise) (CRC Press, Inc., Boca Raton, 1984), pp. 115-138; Hammerling et al., "Monoclonal Antibodies and T-Cell Hybridomas ", Research Monographs in Immunology , Volume 3 (JLTurk, Editor-in-Chief) (Elsevier, New York, 1981), pp. 563-587; Harlow et al., Antibodies: A Laboratory Manual , (Cold Spring Harbor Lab oratory Press, 2nd edition, 1988); Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987)); Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest , 5th Edition, USDepartment of Health and Human Services, NIH Publication Number: 91-3242; Edited by Kontermann and Dübel, Antibody Engineering (2001) Springer-Verlag. New York. Page 790 (ISBN 3-540-41354-5) Kriegler, Gene Transfer and Expression, A Laboratory Manual , Stockton Press, NY (1990); Edited by Lu and Weiner, Cloning and Expression Vectors for Gene Function Analysis (2001) BioTechniques Press. Westborough, Mass. P. 298 (ISBN 1- 881299-21-X); Goodson, JM, Medical Applications of Controlled Release , (ed. By Langer and Wise) (CRC Press, Boca Raton, 1974); Old and Primrose, Principles of Gene Manipulation: An Introduction To Genetic Engineering (No. 3 Edition, 1985) Blackwell Scientific Publications, Boston; Studies in Microbiology , Volume 2: Page 409 (ISBN 0-632-01318-4): Sambrook, J. et al., Molecul ar Cloning: A Laboratory Manual (2nd edition, 1989) Cold Spring Harbor Laboratory Press, NY. Volumes 1-3 (ISBN 0-87969-309-6); Sustained and Controlled Release Drug Delivery Systems , (edited by JR Robinson) ( Marcel Dekker, Inc., New York, 1978); Winnacker, EL From Genes To Clones: Introduction To Gene Technology (1987) VCH Publishers, NY (translated by Horst Ibelgaufts), p. 634 (ISBN 0-89573-614-4 ).

The contents of all references (including references, patents, patent applications, and websites) cited throughout this application are hereby expressly incorporated herein by reference in their entirety, as are the references cited therein. Unless otherwise stated, the practice of the present invention will employ conventional techniques of immunology, molecular biology, and cell biology well known in the art.

Equivalent form

The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. Therefore, the above-described embodiments are to be considered in all respects as illustrative and not restrictive of the invention described herein. Therefore, the scope of the present invention is indicated by the scope of the accompanying patent application rather than by the above description, and therefore all variations that fall within the meaning and range of equivalency of the scope of patent application are intended to be included herein.

<110> American Abbott Corporation

<120> IL-1 binding protein

<130> ABT-121.2 TW

<140> 100116951

<141> 2001-05-13

<150> 61 / 425,701

<151> 2010-12-21

<150> 61 / 334,917

<151> 2010-05-14

<160> 381

<170> PatentIn version 3.5

<210> 1

<211> 159

<212> PRT

<213> Homo sapiens

<400> 1

<210> 2

<211> 153

<212> PRT

<213> Homo sapiens

<400> 2

<210> 3

<211> 330

<212> PRT

<213> Homo sapiens

<400> 3

<210> 4

<211> 330

<212> PRT

<213> Homo sapiens

<400> 4

<210> 5

<211> 106

<212> PRT

<213> Homo sapiens

<400> 5

<210> 6

<211> 105

<212> PRT

<213> Homo sapiens

<400> 6

<210> 7

<211> 30

<212> PRT

<213> Homo sapiens

<400> 7

<210> 8

<211> 14

<212> PRT

<213> Homo sapiens

<400> 8

<210> 9

<211> 32

<212> PRT

<213> Homo sapiens

<400> 9

<210> 10

<211> 11

<212> PRT

<213> Homo sapiens

<400> 10

<210> 11

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<220>

<221> MOD_RES

<222> (3) .. (3)

<223> Any amino acid

<400> 11

<210> 12

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<220>

<221> MOD_RES

<222> (2) .. (9)

<223> Any amino acid

<400> 12

<210> 13

<211> 23

<212> PRT

<213> Homo sapiens

<400> 13

<210> 14

<211> 15

<212> PRT

<213> Homo sapiens

<400> 14

<210> 15

<211> 32

<212> PRT

<213> Homo sapiens

<400> 15

<210> 16

<211> 10

<212> PRT

<213> Homo sapiens

<400> 16

<210> 17

<211> 5

<212> PRT

<213> Homo sapiens

<400> 17

<210> 18

<211> 17

<212> PRT

<213> Homo sapiens

<400> 18

<210> 19

<211> 10

<212> PRT

<213> Homo sapiens

<400> 19

<210> 20

<211> 11

<212> PRT

<213> Homo sapiens

<400> 20

<210> 21

<211> 7

<212> PRT

<213> Homo sapiens

<400> 21

<210> 22

<211> 9

<212> PRT

<213> Homo sapiens

<400> 22

<210> 23

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 23

<210> 24

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<220>

<221> MOD_RES

<222> (3) .. (3)

<223> Any amino acid

<400> 24

<210> 25

<211> 23

<212> PRT

<213> Homo sapiens

<400> 25

<210> 26

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 26

<210> 27

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 27

<210> 28

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 28

<210> 29

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 29

<210> 30

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 30

<210> 31

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 31

<210> 32

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 32

<210> 33

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 33

<210> 34

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 34

<210> 35

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 35

<210> 36

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 36

<210> 37

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 37

<210> 38

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 38

<210> 39

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 39

<210> 40

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 40

<210> 41

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 41

<210> 42

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 42

<210> 43

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 43

<210> 44

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 44

<210> 45

<211> 27

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 45

<210> 46

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 46

<210> 47

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 47

<210> 48

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 48

<210> 49

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 49

<210> 50

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 50

<210> 51

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 51

<210> 52

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 52

<210> 53

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 53

<210> 54

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 54

<210> 55

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 55

<210> 56

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 56

<210> 57

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 57

<210> 58

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 58

<210> 59

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 59

<210> 60

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 60

<210> 61

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 61

<210> 62

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 62

<210> 63

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 63

<210> 64

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 64

<210> 65

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 65

<210> 66

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 66

<210> 67

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 67

<210> 68

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 68

<210> 69

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 69

<210> 70

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 70

<210> 71

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 71

<210> 72

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 72

<210> 73

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 73

<210> 74

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 74

<210> 75

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 75

<210> 76

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 76

<210> 77

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 77

<210> 78

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 78

<210> 79

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 79

<210> 80

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 80

<210> 81

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 81

<210> 82

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 82

<210> 83

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 83

<210> 84

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 84

<210> 85

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 85

<210> 86

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 86

<210> 87

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 87

<210> 88

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 88

<210> 89

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 89

<210> 90

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 90

<210> 91

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 91

<210> 92

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 92

<210> 93

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 93

<210> 94

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 94

<210> 95

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 95

<210> 96

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 96

<210> 97

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 97

<210> 98

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 98

<210> 99

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 99

<210> 100

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 100

<210> 101

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 101

<210> 102

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 102

<210> 103

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 103

<210> 104

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 104

<210> 105

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 105

<210> 106

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 106

<210> 107

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 107

<210> 108

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 108

<210> 109

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 109

<210> 110

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 110

<210> 111

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 111

<210> 112

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 112

<210> 113

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 113

<210> 114

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 114

<210> 115

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 115

<210> 116

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 116

<210> 117

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 117

<210> 118

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 118

<210> 119

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 119

<210> 120

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 120

<210> 121

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 121

<210> 122

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 122

<210> 123

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 123

<210> 124

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 124

<210> 125

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 125

<210> 126

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 126

<210> 127

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 127

<210> 128

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 128

<210> 129

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 129

<210> 130

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 130

<210> 131

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 131

<210> 132

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 132

<210> 133

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 133

<210> 134

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 134

<210> 135

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 135

<210> 136

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 136

<210> 137

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 137

<210> 138

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 138

<210> 139

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 139

<210> 140

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 140

<210> 141

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 141

<210> 142

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 142

<210> 143

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 143

<210> 144

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 144

<210> 145

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 145

<210> 146

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 146

<210> 147

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 147

<210> 148

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 148

<210> 149

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 149

<210> 150

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 150

<210> 151

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 151

<210> 152

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 152

<210> 153

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 153

<210> 154

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 154

<210> 155

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 155

<210> 156

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 156

<210> 157

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 157

<210> 158

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 158

<210> 159

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 159

<210> 160

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 160

<210> 161

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 161

<210> 162

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 162

<210> 163

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 163

<210> 164

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 164

<210> 165

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 165

<210> 166

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 166

<210> 167

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 167

<210> 168

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 168

<210> 169

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 169

<210> 170

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 170

<210> 171

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 171

<210> 172

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 172

<210> 173

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 173

<210> 174

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 174

<210> 175

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 175

<210> 176

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 176

<210> 177

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 177

<210> 178

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 178

<210> 179

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 179

<210> 180

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 180

<210> 181

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 181

<210> 182

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 182

<210> 183

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 183

<210> 184

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 184

<210> 185

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 185

<210> 186

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 186

<210> 187

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 187

<210> 188

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 188

<210> 189

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 189

<210> 190

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<220>

<221> MOD_RES

<222> (1) .. (1)

<223> Ser, Lys or Arg

<400> 190

<210> 191

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> Ile or Val

<220>

<221> MOD_RES

<222> (4) .. (4)

<223> Ser or His

<220>

<221> MOD_RES

<222> (7) .. (7)

<223> Gly or Ala

<220>

<221> MOD_RES

<222> (14) .. (14)

<223> Thr or Ser

<220>

<221> MOD_RES

<222> (15) .. (15)

<223> Val or Ala

<400> 191

<210> 192

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<220>

<221> MOD_RES

<222> (4) .. (4)

<223> Thr or Tyr

<220>

<221> MOD_RES

<222> (7) .. (7)

<223> Tyr or Cys

<220>

<221> MOD_RES

<222> (10) .. (10)

<223> Val, Glu, Leu, Met, Gln, or Tyr

<400> 192

<210> 193

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<220>

<221> MOD_RES

<222> (7) .. (7)

<223> His, Tyr or Trp

<220>

<221> MOD_RES

<222> (8) .. (8)

<223> Asn, Gly, Thr, Gln, Glu, His, Asp, or Lys

<220>

<221> MOD_RES

<222> (9) .. (9)

<223> Tyr or Trp

<220>

<221> MOD_RES

<222> (11) .. (11)

<223> Thr, Ala, or Asn

<400> 193

<210> 194

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<220>

<221> MOD_RES

<222> (1) .. (1)

<223> Asn, Gln, or Asp

<220>

<221> MOD_RES

<222> (4) .. (4)

<223> Thr, Asn, Ile, Glu or Ser

<220>

<221> MOD_RES

<222> (6) .. (6)

<223> Ala, Met or Glu

<220>

<221> MOD_RES

<222> (7) .. (7)

<223> Asp, Glu, Ser or Ala

<400> 194

<210> 195

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<220>

<221> MOD_RES

<222> (2) .. (2)

<223> His or Gln

<220>

<221> MOD_RES

<222> (5) .. (5)

<223> Ser, Asn, Thr, Lys, Arg or Met

<220>

<221> MOD_RES

<222> (6) .. (6)

<223> Ile or Leu

<220>

<221> MOD_RES

<222> (8) .. (8)

<223> Tyr or Ala

<220>

<221> MOD_RES

<222> (9) .. (9)

<223> Thr, Ile, or Asn

<400> 195

<210> 196

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 196

<210> 197

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 197

<210> 198

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 198

<210> 199

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 199

<210> 200

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 200

<210> 201

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 201

<210> 202

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 202

<210> 203

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 203

<210> 204

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 204

<210> 205

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 205

<210> 206

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 206

<210> 207

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 207

<210> 208

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 208

<210> 209

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 209

<210> 210

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 210

<210> 211

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 211

<210> 212

<211> 247

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 212

<210> 213

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 213

<210> 214

<211> 330

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 214

<210> 215

<211> 221

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 215

<210> 216

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 216

<210> 217

<211> 254

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 217

<210> 218

<211> 228

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 218

<210> 219

<211> 247

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 219

<210> 220

<211> 221

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 220

<210> 221

<211> 254

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 221

<210> 222

<211> 228

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 222

<210> 223

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 223

<210> 224

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 224

<210> 225

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 225

<210> 226

<211> 247

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 226

<210> 227

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 227

<210> 228

<211> 221

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 228

<210> 229

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 229

<210> 230

<211> 247

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 230

<210> 231

<211> 221

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 231

<210> 232

<211> 247

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 232

<210> 233

<211> 221

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 233

<210> 234

<211> 247

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 234

<210> 235

<211> 221

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 235

<210> 236

<211> 254

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 236

<210> 237

<211> 228

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 237

<210> 238

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 238

<210> 239

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 239

<210> 240

<211> 30

<212> PRT

<213> Homo sapiens

<400> 240

<210> 241

<211> 14

<212> PRT

<213> Homo sapiens

<400> 241

<210> 242

<211> 32

<212> PRT

<213> Homo sapiens

<400> 242

<210> 243

<211> 11

<212> PRT

<213> Homo sapiens

<400> 243

<210> 244

<211> 30

<212> PRT

<213> Homo sapiens

<400> 244

<210> 245

<211> 14

<212> PRT

<213> Homo sapiens

<400> 245

<210> 246

<211> 32

<212> PRT

<213> Homo sapiens

<400> 246

<210> 247

<211> 11

<212> PRT

<213> Homo sapiens

<400> 247

<210> 248

<211> 30

<212> PRT

<213> Homo sapiens

<400> 248

<210> 249

<211> 14

<212> PRT

<213> Homo sapiens

<400> 249

<210> 250

<211> 32

<212> PRT

<213> Homo sapiens

<400> 250

<210> 251

<211> 11

<212> PRT

<213> Homo sapiens

<400> 251

<210> 252

<211> 30

<212> PRT

<213> Homo sapiens

<400> 252

<210> 253

<211> 14

<212> PRT

<213> Homo sapiens

<400> 253

<210> 254

<211> 32

<212> PRT

<213> Homo sapiens

<400> 254

<210> 255

<211> 11

<212> PRT

<213> Homo sapiens

<400> 255

<210> 256

<211> 30

<212> PRT

<213> Homo sapiens

<400> 256

<210> 257

<211> 14

<212> PRT

<213> Homo sapiens

<400> 257

<210> 258

<211> 32

<212> PRT

<213> Homo sapiens

<400> 258

<210> 259

<211> 11

<212> PRT

<213> Homo sapiens

<400> 259

<210> 260

<211> 30

<212> PRT

<213> Homo sapiens

<400> 260

<210> 261

<211> 14

<212> PRT

<213> Homo sapiens

<400> 261

<210> 262

<211> 32

<212> PRT

<213> Homo sapiens

<400> 262

<210> 263

<211> 11

<212> PRT

<213> Homo sapiens

<400> 263

<210> 264

<211> 30

<212> PRT

<213> Homo sapiens

<400> 264

<210> 265

<211> 14

<212> PRT

<213> Homo sapiens

<400> 265

<210> 266

<211> 32

<212> PRT

<213> Homo sapiens

<400> 266

<210> 267

<211> 11

<212> PRT

<213> Homo sapiens

<400> 267

<210> 268

<211> 30

<212> PRT

<213> Homo sapiens

<400> 268

<210> 269

<211> 14

<212> PRT

<213> Homo sapiens

<400> 269

<210> 270

<211> 32

<212> PRT

<213> Homo sapiens

<400> 270

<210> 271

<211> 11

<212> PRT

<213> Homo sapiens

<400> 271

<210> 272

<211> 30

<212> PRT

<213> Homo sapiens

<400> 272

<210> 273

<211> 14

<212> PRT

<213> Homo sapiens

<400> 273

<210> 274

<211> 32

<212> PRT

<213> Homo sapiens

<400> 274

<210> 275

<211> 11

<212> PRT

<213> Homo sapiens

<400> 275

<210> 276

<211> 30

<212> PRT

<213> Homo sapiens

<400> 276

<210> 277

<211> 14

<212> PRT

<213> Homo sapiens

<400> 277

<210> 278

<211> 32

<212> PRT

<213> Homo sapiens

<400> 278

<210> 279

<211> 11

<212> PRT

<213> Homo sapiens

<400> 279

<210> 280

<211> 30

<212> PRT

<213> Homo sapiens

<400> 280

<210> 281

<211> 14

<212> PRT

<213> Homo sapiens

<400> 281

<210> 282

<211> 32

<212> PRT

<213> Homo sapiens

<400> 282

<210> 283

<211> 11

<212> PRT

<213> Homo sapiens

<400> 283

<210> 284

<211> 30

<212> PRT

<213> Homo sapiens

<400> 284

<210> 285

<211> 14

<212> PRT

<213> Homo sapiens

<400> 285

<210> 286

<211> 32

<212> PRT

<213> Homo sapiens

<400> 286

<210> 287

<211> 11

<212> PRT

<213> Homo sapiens

<400> 287

<210> 288

<211> 30

<212> PRT

<213> Homo sapiens

<400> 288

<210> 289

<211> 14

<212> PRT

<213> Homo sapiens

<400> 289

<210> 290

<211> 32

<212> PRT

<213> Homo sapiens

<400> 290

<210> 291

<211> 11

<212> PRT

<213> Homo sapiens

<400> 291

<210> 292

<211> 30

<212> PRT

<213> Homo sapiens

<400> 292

<210> 293

<211> 14

<212> PRT

<213> Homo sapiens

<400> 293

<210> 294

<211> 32

<212> PRT

<213> Homo sapiens

<400> 294

<210> 295

<211> 11

<212> PRT

<213> Homo sapiens

<400> 295

<210> 296

<211> 30

<212> PRT

<213> Homo sapiens

<400> 296

<210> 297

<211> 14

<212> PRT

<213> Homo sapiens

<400> 297

<210> 298

<211> 32

<212> PRT

<213> Homo sapiens

<400> 298

<210> 299

<211> 11

<212> PRT

<213> Homo sapiens

<400> 299

<210> 300

<211> 30

<212> PRT

<213> Homo sapiens

<400> 300

<210> 301

<211> 14

<212> PRT

<213> Homo sapiens

<400> 301

<210> 302

<211> 32

<212> PRT

<213> Homo sapiens

<400> 302

<210> 303

<211> 11

<212> PRT

<213> Homo sapiens

<400> 303

<210> 304

<211> 30

<212> PRT

<213> Homo sapiens

<400> 304

<210> 305

<211> 14

<212> PRT

<213> Homo sapiens

<400> 305

<210> 306

<211> 32

<212> PRT

<213> Homo sapiens

<400> 306

<210> 307

<211> 11

<212> PRT

<213> Homo sapiens

<400> 307

<210> 308

<211> 30

<212> PRT

<213> Homo sapiens

<400> 308

<210> 309

<211> 14

<212> PRT

<213> Homo sapiens

<400> 309

<210> 310

<211> 32

<212> PRT

<213> Homo sapiens

<400> 310

<210> 311

<211> 11

<212> PRT

<213> Homo sapiens

<400> 311

<210> 312

<211> 11

<212> PRT

<213> Homo sapiens

<400> 312

<210> 313

<211> 30

<212> PRT

<213> Homo sapiens

<400> 313

<210> 314

<211> 14

<212> PRT

<213> Homo sapiens

<400> 314

<210> 315

<211> 32

<212> PRT

<213> Homo sapiens

<400> 315

<210> 316

<211> 11

<212> PRT

<213> Homo sapiens

<400> 316

<210> 317

<211> 15

<212> PRT

<213> Homo sapiens

<400> 317

<210> 318

<211> 32

<212> PRT

<213> Homo sapiens

<400> 318

<210> 319

<211> 10

<212> PRT

<213> Homo sapiens

<400> 319

<210> 320

<211> 23

<212> PRT

<213> Homo sapiens

<400> 320

<210> 321

<211> 15

<212> PRT

<213> Homo sapiens

<400> 321

<210> 322

<211> 32

<212> PRT

<213> Homo sapiens

<400> 322

<210> 323

<211> 10

<212> PRT

<213> Homo sapiens

<400> 323

<210> 324

<211> 23

<212> PRT

<213> Homo sapiens

<400> 324

<210> 325

<211> 15

<212> PRT

<213> Homo sapiens

<400> 325

<210> 326

<211> 32

<212> PRT

<213> Homo sapiens

<400> 326

<210> 327

<211> 11

<212> PRT

<213> Homo sapiens

<400> 327

<210> 328

<211> 23

<212> PRT

<213> Homo sapiens

<400> 328

<210> 329

<211> 15

<212> PRT

<213> Homo sapiens

<400> 329

<210> 330

<211> 32

<212> PRT

<213> Homo sapiens

<400> 330

<210> 331

<211> 11

<212> PRT

<213> Homo sapiens

<400> 331

<210> 332

<211> 23

<212> PRT

<213> Homo sapiens

<400> 332

<210> 333

<211> 15

<212> PRT

<213> Homo sapiens

<400> 333

<210> 334

<211> 32

<212> PRT

<213> Homo sapiens

<400> 334

<210> 335

<211> 11

<212> PRT

<213> Homo sapiens

<400> 335

<210> 336

<211> 23

<212> PRT

<213> Homo sapiens

<400> 336

<210> 337

<211> 15

<212> PRT

<213> Homo sapiens

<400> 337

<210> 338

<211> 32

<212> PRT

<213> Homo sapiens

<400> 338

<210> 339

<211> 11

<212> PRT

<213> Homo sapiens

<400> 339

<210> 340

<211> 22

<212> PRT

<213> Homo sapiens

<400> 340

<210> 341

<211> 15

<212> PRT

<213> Homo sapiens

<400> 341

<210> 342

<211> 32

<212> PRT

<213> Homo sapiens

<400> 342

<210> 343

<211> 10

<212> PRT

<213> Homo sapiens

<400> 343

<210> 344

<211> 22

<212> PRT

<213> Homo sapiens

<400> 344

<210> 345

<211> 15

<212> PRT

<213> Homo sapiens

<400> 345

<210> 346

<211> 32

<212> PRT

<213> Homo sapiens

<400> 346

<210> 347

<211> 10

<212> PRT

<213> Homo sapiens

<400> 347

<210> 348

<211> 21

<212> PRT

<213> Homo sapiens

<400> 348

<210> 349

<211> 15

<212> PRT

<213> Homo sapiens

<400> 349

<210> 350

<211> 32

<212> PRT

<213> Homo sapiens

<400> 350

<210> 351

<211> 10

<212> PRT

<213> Homo sapiens

<400> 351

<210> 352

<211> 22

<212> PRT

<213> Homo sapiens

<400> 352

<210> 353

<211> 15

<212> PRT

<213> Homo sapiens

<400> 353

<210> 354

<211> 32

<212> PRT

<213> Homo sapiens

<400> 354

<210> 355

<211> 11

<212> PRT

<213> Homo sapiens

<400> 355

<210> 356

<211> 23

<212> PRT

<213> Homo sapiens

<400> 356

<210> 357

<211> 15

<212> PRT

<213> Homo sapiens

<400> 357

<210> 358

<211> 32

<212> PRT

<213> Homo sapiens

<400> 358

<210> 359

<211> 11

<212> PRT

<213> Homo sapiens

<400> 359

<210> 360

<211> 23

<212> PRT

<213> Homo sapiens

<400> 360

<210> 361

<211> 15

<212> PRT

<213> Homo sapiens

<400> 361

<210> 362

<211> 32

<212> PRT

<213> Homo sapiens

<400> 362

<210> 363

<211> 11

<212> PRT

<213> Homo sapiens

<400> 363

<210> 364

<211> 23

<212> PRT

<213> Homo sapiens

<400> 364

<210> 365

<211> 15

<212> PRT

<213> Homo sapiens

<400> 365

<210> 366

<211> 32

<212> PRT

<213> Homo sapiens

<400> 366

<210> 367

<211> 11

<212> PRT

<213> Homo sapiens

<400> 367

<210> 368

<211> 22

<212> PRT

<213> Homo sapiens

<400> 368

<210> 369

<211> 15

<212> PRT

<213> Homo sapiens

<400> 369

<210> 370

<211> 32

<212> PRT

<213> Homo sapiens

<400> 370

<210> 371

<211> 10

<212> PRT

<213> Homo sapiens

<400> 371

<210> 372

<211> 22

<212> PRT

<213> Homo sapiens

<400> 372

<210> 373

<211> 15

<212> PRT

<213> Homo sapiens

<400> 373

<210> 374

<211> 32

<212> PRT

<213> Homo sapiens

<400> 374

<210> 375

<211> 10

<212> PRT

<213> Homo sapiens

<400> 375

<210> 376

<211> 23

<212> PRT

<213> Homo sapiens

<400> 376

<210> 377

<211> 15

<212> PRT

<213> Homo sapiens

<400> 377

<210> 378

<211> 32

<212> PRT

<213> Homo sapiens

<400> 378

<210> 379

<211> 11

<212> PRT

<213> Homo sapiens

<400> 379

<210> 380

<211> 11

<212> PRT

<213> Homo sapiens

<400> 380

<210> 381

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptides

<400> 381

Claims (17)

A binding protein comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a first VD1-X1-VD2-C-X2, wherein: VD1 is a first heavy chain variable domain; VD2 is the second heavy chain variable domain; C is the heavy chain constant domain; X1 is the linker, with the limitation that it is not CH1; and X2 is the Fc region; and wherein the second polypeptide chain contains the second VD1-X1- VD2-C, where: VD1 is the first light chain variable domain; VD2 is the second light chain variable domain; C is the light chain constant domain; and X1 is a linker with the restriction that it is not CH1; and wherein, In the first polypeptide chain, VD1 includes the amino acid sequence of SEQ ID NO: 204; X1 includes the amino acid sequence of SEQ ID NO: 33; VD2 includes the amino acid sequence of SEQ ID NO: 213; and the The heavy chain constant domain is an IgG1 heavy chain constant region comprising a mutation in the hinge region; wherein, in the second polypeptide chain, VD1 comprises the amino acid sequence of SEQ ID NO: 238; X1 comprises the amino group of SEQ ID NO: 35 Acid sequence; and VD2 comprises the amino acid sequence of SEQ ID NO: 216; and wherein the binding protein binds human IL-1β and human IL-1α. A binding protein comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a first VD1-X1-VD2-C-X2, wherein: VD1 is the variable domain of the first heavy chain; VD2 is the variable domain of the second heavy chain; C is the constant domain of the heavy chain; X1 is a linker with the restriction that it is not CH1; and X2 is the Fc region; and wherein the second The polypeptide chain includes a second VD1-X1-VD2-C, where: VD1 is the first light chain variable domain; VD2 is the second light chain variable domain; C is the light chain constant domain; and X1 is a linker, limiting Provided that it is not CH1; and is characterized in that VD1-X1-VD2 in the first polypeptide chain is SEQ ID NO: 212, and C-X2 in the first polypeptide chain is SEQ ID NO: 214 ; VD1-X1-VD2 in the second polypeptide chain is SEQ ID NO: 215, and C in the second polypeptide chain is SEQ ID NO: 5; and wherein the binding protein binds human IL-1β and human IL-1α. The binding protein of claim 1, wherein the hinge region mutation comprises L234A. The binding protein of claim 1, wherein the hinge region mutation comprises L235A. The binding protein of claim 1, wherein the hinge region mutation comprises L234A and L235A. The binding protein according to any one of claims 1 to 5, characterized in that the binding protein comprises two first polypeptide chains and two second polypeptide chains. A pharmaceutical composition comprising a binding protein according to any one of claims 1 to 6, and a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 7, further comprising at least one other agent. The pharmaceutical composition of claim 8, wherein the other agent is selected from the group consisting of Group: therapeutic agents; imaging agents; cytotoxic agents; angiogenesis inhibitors; kinase inhibitors; costimulatory molecule blockers; adhesion molecule blockers; anti-cytokine antibodies or functional fragments thereof; methotrexate Cyclosporin; rapamycin; FK506; detectable markers or reporters; TNF antagonists; antirheumatic drugs; muscle relaxants; anesthetics; nonsteroidal anti-inflammatory drugs (NSAID); analgesics Agents; narcotics; sedatives; local anaesthetics; neuromuscular blocking agents; antimicrobials; anti-psoriasis drugs; corticosteroids; anabolic steroids; erythropoietin; immunity; immunoglobulins; immunosuppressants; Growth hormones; hormone replacement drugs; radiopharmaceuticals; antidepressants; antipsychotics; stimulants; asthma drugs; beta agonists; inhaled steroids; epinephrine or its analogs; cytokines; and cytokine antagonists. An isolated nucleic acid comprising a binding protein as set forth in any one of claims 1 to 6. A vector comprising an isolated nucleic acid as claimed in claim 10. A host cell comprising a vector as claimed in claim 11. A method for preparing a binding protein comprising culturing a host cell as claimed in claim 12 in a medium under conditions sufficient to produce the binding protein. An in vitro method for reducing human IL-1 activity, comprising contacting a human IL-1 protein with a binding protein according to any one of claims 1 to 6 in order to reduce human IL-1 protein activity. Use of a binding protein according to any one of claims 1 to 6 for the manufacture of a medicament for reducing human IL-1 activity in a human individual suffering from a condition in which IL-1 activity is harmful, wherein the condition is inflammation Illness. A use of a binding protein according to any one of claims 1 to 6 for the manufacture of a medicament for treating a condition in which IL-1 activity of an individual is harmful, wherein the condition is an inflammatory condition. The use according to claim 16, wherein the disorder is selected from the group consisting of rheumatoid arthritis; osteoarthritis; juvenile chronic arthritis; septic arthritis; Lyme arthritis; psoriasis Arthritis and reactive arthritis.